الفهرس 
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Abstract
Many Mycoplasma species are important veterinary pathogens causing many different diseases and occasionally abortion in farm animals. This study focused on both M. bovis and M. bovigenitlium as they are among the most important Mycoplasma species.
The aim of this study was to apply and evaluate the use of a general Mycoplasma specific PCR assay for detection of Mycoplasma in clinical samples. Moreover, identification of specifically M. bovis and M. bovigenitalium in positive clinical samples will be performed. Also, comparison between results of the applied PCR assays and conventional methods for isolation and identification will be performed.
Samples used in this study were collected from different farms in Egypt. The samples were 137 cow’s individual milk, 62 cow’s bulk tank milk, 44 bovine frozen semen and 10 uterine secretions of aborted cows and 4 aborted foeti samples.
Based on cultural, morphological and biochemical characters, 21 bacterial isolates identified as Mycoplasma bovis were recovered from 257 samples in a percentage of 8.2 %.
Among the 21 Mycoplasma isolates recovered from total 257 sample, 16 isolates were recovered from 137 milk samples in a percentage of 11.68%, 3 isolates were recovered from 62 bulk tank milk in a percentage of 4.8 %, 1 isolate was recovered from 10 fetal fluids and 1 isolate was recovered from 4 aborted foeti in percentages of 10 and 25%, respectively.
The overall percentage of Mycoplasma positive clinical samples using PCR was 37.35% which was very interesting where this percentage was very high when compared to that obtained by using conventional diagnostic methods (8.2%).
General Mycoplasma PCR revealed that 96 samples successfully amplified the 270 bp PCR products and therefore were considered positive for Mycoplasma species.
The PCR positive samples were as follow, 69/137 milk samples in a percentage of 50.4%, 12/62 bulk tank milk in a percentage of 19.35 %, 8/10 fetal fluids samples in a percentage of 80% and 3/4 aborted foetus samples in a percentage of 75% and 4/44 frozen semen samples in a percentage of 9.1 %.
The other aim of this study was the identification of Mycoplasma directly in clinical samples. Therefore, molecular identification was carried out by subjecting the positive samples for both Mycoplasma bovis and Mycoplasma bovigenitallium specific PCR assays. Out of the 96 Mycoplasma positive samples, 73 samples were found to contain Mycoplasma bovis (227 bp positive) while 11 samples were found to contain Mycoplasma bovigenitallium (310 bp positive). Ten Mycoplasma positive samples contained mixed infection of both M. bovis and M. bovigenitallium. Moreover, 2 positive samples were negative for both M. bovis and M. bovigenitalium and therefore supposed to contain other species of Mycoplasma.
Comparison between results of the applied PCR assays and conventional methods for isolation and identification revealed clearly that the PCR could identify more positive samples beside its rapidity when compared with conventional methods.
Using Real time PCR, there was no discrepent results between results of both PCR and Real time PCR among the tested Mycoplasma strains and isolates. Considering its use for detection of Mycoplasma in milk samples (29), only 14 samples were positive versus 27 positive by conventional PCR. So, QPCR requires more optimization trials including the best DNA extraction methods.
In conclusion, the molecular approach for detection and identification of Mycoplasma species directly in clinical samples can be better alternatives to the traditional approach. It was found to be sensitive, straightforward and fast tool for Mycoplasma detection in different clinical samples especially from samples difficult to be cultured.