Untersuchungen zur Prävalenz von Mycoplasma suis in Deutschland sowie vergleichende Untersuchungen zwischen real-time Polymerasekettenreaktion und Akridin-Ausstrich bezüglich ihrer Sensitivität und Spezifität

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.