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Grimm, Julia (2008): Untersuchungen zur Prävalenz von Mycoplasma suis in Deutschland sowie vergleichende Untersuchungen zwischen real-time Polymerasekettenreaktion und Akridin-Ausstrich bezüglich ihrer Sensitivität und Spezifität. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

Investigation of the distribution of Mycoplasma suis and comparing investigations of real-time PCR and acridine orange-stained blood smears concerning sensitivity and specificity As the porcine Eperythrozoonosis is most commonly a latent chronic infection, a high sensitive assay is required to evaluate the distribution of Mycoplasma suis in Germany. Due to its high sensitivity and specificity the real-time PCR was chosen for this study. In comparison to conventional PCR assays the real-time PCR is a rapid, easy-doing, more accurate and more sensitive system. In the present study the prevalence of M. suis in Germany was measured by using the LightCycler® System and a constant real-time PCR-protocol. Additionally a new method of DNA-Extraction, the Gen Elute Bacterial Genomic DNA Kit, was evaluated during this study. The Gen Elute Bacterial Genomic DNA Kit showed the same results concerning sensitivity and efficiency of DNA extraction as the automatical extraction with the MagNA Pure LCTM System. 1176 blood samples of slaughtered post-weaning pigs from 196 different pig herds were collected. In addition to PCR analysis a haemogram was made from any sample and clinicochemical parameter were determined. Moreover an acridine orange-stained blood smear was analysed from each sample. Real-time PCR showed a positive result for 164 out of 1176 samples (13,9%). With the acridine stain only 35 pigs were identified as infected with M. suis. 31 of these samples were also positive in the real-time PCR. In the present study microscopic examination on stained blood smears only detected M. suis infections with a bacterial load of at least 105 per ml blood. 80 (40,8%) out of 196 pig herds were detected positive for M .suis by real-time PCR. The number of M. suis infected herds in the different districts of Germany varied from 33,3% to 48%. The prevalence within one herd varried from 25% to 46,2% and showed an average value of 34,2%. A significant correlation between the bacterial load per ml blood and the degree of severity of anemia was shown. A decrease of the number of erythrocytes, hemoglobin concentration and hematocrit was observed when the bacterial load increased. By comparing real-time PCR and microscopic examination of acridine orange-stained blood smears it was shown that the real-time PCR system is able to detect even latent M. suis infections that are missed out in the microscopic examination. Furthermore immature erythrocytes and Howell-Jolly-bodies may lead to false positive results. With the use of the M. suis specific hybridisation probe system in the real-time PCR false positive results can be avoided. The LightCycler® MSG1 protocol has proven to be a high sensitive and easy-doing system that allows the integration in routine laboratories.