Veterinary Research Communications, 28 (2004) 279–282 © 2004 Kluwer Academic Publishers. Printed in the Netherlands
Improved Detection of Coxiella burnetii in Cows Milk by Immunomagnetic Separation and PCR F. Capuano1*, A.G. Perugini1, A. Parisi2, C.O. Montagna2 and M. Nilvelli1 1Istituto Zooprofilattico Sperimentale del Mezzogiorno, sez. di Avellino; 2Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, sez. di Bari *Correspondence: Istituto Zooprofilattico Sperimentale del Mezzogiorno, L aboratorio di Biotecnologie, via Salute 2, 80055 Portici, Napoli, Italy E-mail:
[email protected] Keywords: Coxiella burnetii, cows milk, immunomagnetic separation, PCR Abbreviations: IMS, immunomagnetic separation; PCR, polymerase chain reaction
INTRODUCTION The serum prevalence of Coxiella burnetii found in herds tested in the South of Italy (Landolfi et al., 2000; Capuano et al., 2001; Capuano et al., 2002) encouraged us to improve the performance of the PCR test to detect the C. burnetii pathogen in milk.
MATERIALS AND METHODS Setting cow milk samples: one cow milk sample of each group was contaminated in activated cells infected with Coxiella burnetii (Dade Behring, Marburg GmbH, Germany). Each sample was serially diluted to ascertain the reliability of PCR, combined with other different techniques. After contamination, group I specimens were packed by centrifugation at room temperature at 2000 G for 20 min. All the pellets were washed three times with PBS (phosphate-buffered saline, pH 7.4) and resuspended in PBS. To detect DNA C. burnetii from group II specimens, the positive serum for Q fever (Dade Behring, Marburg GmbH, Germany) was diluted 1:1000 and added to artificially infected milk specimens. After one hour of incubation at room temperature, all samples were centrifuged at 10,000 G for 10 min, washed three times and resuspended in PBS. Immunomagnetic beads (IMB) covered with sheep antibody against rabbit IgG were added to the immunoprecipitates (Dynabeads M280) at the final concentration of 1.2×108 beads/ml. Lastly, the specimens were determined by rotation for two hours at room temperature (Muramatsu et al., 1996). The IMB belonging to group II samples were caught by trapping them against the surface of the Eppendorff like tube, using a magnetic particle concentrator (MCP-M, 279
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DYNAL A.S.): then after removing the surplus fluid, all IMB were washed three times with PBS and lastly, resuspended in PBS. Group III specimens were prepared for group II and then IMB was added and the samples were rotated for 18 h at 4°C to promote interaction between all the immunoprecipitates and the antibodies covering the IMB. Before addition at the usual concentration to the group IV samples, all IMB were rotated for 18 h at 4°C after addition of the positive serum for C. burnetii. After the addition of IMB, the group IV specimens were rotated for two hours at room temperature and they were then determined as for those of III group to collect all the IMB. All the precipitates, resuspended in PBS, were boiled for 10 min and one portion of each sample was used for PCR assay.
PCR PCR was performed according to the method designed for the amplification of htpAB gene and one repeated region of the C. burnetii genome by Berri et al. (2000) using Trans-1 and Trans-2 primers (Fournier et al., 1998).
RESULTS We did not observe any non-specific amplification fragments in each group, although the direct IMB (group IV – Fig. 3) was the most reliable and rapid for detection of the pathogen C. burnetii as compared with classical and indirect (group II – Fig. 1) or modified IMS (group III – Fig. 2) methods.
Figure 1. Group II (indirect IMS) – PCR. L: ladder 100 bp (Genenco, Life-Science, Milan). Coxiella serially diluted in milk: 1=10−2; 2=10−5; 3=10−8; 4=10−11; 5=10−14; 6=10−17, 7=10−20
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Figure 2. Group III (indirect and modified IMS) – PCR. L: ladder 100 bp (Genenco, LifeScience, Milan). Coxiella serially diluted in milk: 1=10−2; 2=10−5; 3=10−8; 4=10−11; 5=10−14; 6=10−17, 7=10−20
Figure 3. Group IV (direct IMS) – PCR. L: ladder 100 bp (Genenco, Life-Science, Milan). Coxiella serially diluted in milk: 1=10−2; 2=10−5; 3=10−8; 4=10−11; 5=10−14; 6=10−17, 7=10−20
DISCUSSION The PCR test is very useful for the detection of C. burnetii in milk, but it is not always a diagnostically sensitive and reliable method, especially in milk collections, due to the presence of some substances that inhibit DNA amplification. The right choice of technique for sample preparation is fundamental for the improvement of detection PCR. This study is so important to stress that both indirect and modified IMS and direct IMS are the only diagnostic methods able to detect C. burnetii in fully diluted samples (over 10−11 dilutions) and this may be because of the extended interaction time between IMB and the positive serum for Q fever in these techniques allows determination of the bacterial cells contaminating all the samples. Thus, direct IMS is really the most reliable method to detect C. burnetii by PCR, which is why only five hours are required to observe amplification.
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REFERENCES Berri, M., Laroucau, K. and Rodolakis, A. 2000. The detection of Coxiella from ovin genital swabs, milk and fecal samples by the use of single touchdowm polymerase chain reaction. Veterinary Microbiology, 72, 285–293 Capuano, F., Landolfi, M.C. and Monetti, D.M., 2001. Influence of three types of farm management on the seroprevalence of Q fever as assessed by an indirect immunofluorescence assay. Veterinary Record, 149(22), 669–71 Capuano, F., Landolfi, M.C., Rinaldi, L., Veneziano, V., Capelli, G. and Cringoli, G., 2002. Epidemiological survey of Q fever on dogs in an area of the Southern Italian Appennines and correlations with leishmaniosis and rickettsiosis. Atti Societa` Italiana Scienze Veterinarie, 197–198 Fournier, P.E., Marrie, T.J. and Raoult, D., 1998. Diagnosis of Q fever. Journal of Clinical Microbiology, 36(7), 1823–1834 Landolfi, M.C., Esposito, A., Varlese, M.L., Fiore, E. and Capuano. F. 2000 Atti Societa` Italiana Patologia Allevamento Ovini Caprini, 127–130
