الفهرس | Only 14 pages are availabe for public view |
Abstract Paratuberculosis or PTB or Johne‘s disease is a chronic mammalian disease, it is caused by Mycobacterium avium subsp. paratuberculosis (Map). Johne‘s disease is considered as one of the most serious problems affecting the world‘s ruminant industry due to significant impact on global economy. It resembles some clinical and pathological aspects of Crohn‘s disease in humans. The classical method of diagnosis of PTB takes long incubation period, high costs, and possible contamination, so the aim of this study was to develop fast and sensitive diagnosis technique for detection of Map in faecal and milk samples of the same animal among the Egyptian farms, using different primers and different PCR assays and comparing between them to find the most sensitive method for detecting the infection. In addition, we evaluated the effect of Map infection on cattle milk production. For this, a total sample of 300 consisting of 150 cattle faeces and 150 individual cow‘s milk were collected randomly from the cattle with suspected and health status. The samples were assayed using IS900 and F57 primers. We found that the real-time PCR assay is more sensitive than conventional PCR since it could detect 1.1x10-8 μg/μl pure DNA while the conventional PCR detected 1.1x10-6 μg/μl using IS900 primer, and we also found that the using F57 primer in conventional PCR assay have high sensitivity than IS900 primer since it could detect 1.1x10-13 μg/μl pure DNA. But in clinical samples real-time PCR showed high sensitive in detection of Map DNA, it detected 42% from total 150 faecal samples and the conventional PCR detect 31.33% by using IS900 primer set. And in milk samples real-time detected Map DNA in 5.3%, while the conventional PCR detected 4.67 % of total 150 milk samples, and by comparing the sensitivity of two different primers on clinical samples they have shown the same sensitivity. Where F57 primer detected 28.67% of total 150 faecal samples and 3.33% of total 150 milk samples, showing the same sensitivity with IS900 primer in detecting Map infection in faecal and milk samples. The samples were divided according to the healthy statues of the animals and the results was (34.6%), (32.05%) and (39.7%) of total 77 diseased animals using IS900, F57 conventional PCR and IS900 real-time PCR, respectively and this results have been shown that no significant difference in the detection sensitivity in 98 diseased animals between of two primers (IS900 and F57) and different PCR assays (real-time and conventional PCR) using IS900. But in apparently healthy animals the results was (27.4%), (26.03%) and (45.21%) of total 73 samples using IS900, F57 conventional PCR and IS900 real-time PCR, respectively which appeared a significant different in the sensitivity of the detection of IS900 realtime PCR for Map than IS900 conventional PCR but no difference between IS900 primer set and F57 Primer set. The percentage of infection among the three governorates showed that the highest percentage was in El-Behera then Beni-Suef and the lowest percentage was in El- Dakahlia. In the milk samples the detection limit of the two PCR assays showed no significant difference; also no difference between the two primers that wwere used. But the high significant difference in detecting the infection was shown between the faecal and milk samples. And by taking the history of milk production of the animals we found that the symptomic animals showed decrease in their milk production that ranged from 2.14% to 69.4%, while asymptomic animals ranged from 2% to 25%. The high reduction in milk production was found in El-Behera then in Beni-Suef and then El-Dakahlia. |