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The study aimed at direct detection of Mycobacterium avium subsp. Paratuberculosis (MAP) in milk by evaluating a multiplex real-time PCR assay targeting IS900 and ISMAV2 sequences including the amplification of PUC19-plasmid as internal control. The sensitivity of the assays was evaluated by testing MAP isolates in broad linear range of DNA (50 ng – 5 fg/μl). For the validation of the specificity, 6 MAP isolates and 22 isolates of genus Mycobacteriaceae were tested.Results revealed that reproducible detection limit for real-time PCR targeting IS900 and ISMAV2 was 5 fg/μl and 50 fg/μl respectively. By targeting ISMAV2 sequence, 100% specificity was detected. However, a cross reaction with 5 ng/μl of genome of 3 M. avian subspecies avium strains was detected by targeting IS900 and negative in lower genome quantity (5pg/μl). To maximize the assay’s detection sensitivity,an efficient strategy for MAP-DNA extraction from spiked milk was assessed. Targeting of IS900 was sensitive and targeting ISMAV2 was very specific. Therefore, a multiplex real-time PCR assay targeting IS900 and ISMAV2 in combination with two commercial DNA extraction kits could be an ideal sensitive and specific protocol for routine large-scale analysis of milk samples and other clinical specimens from man and animals.
Paratuberculosis is a chronic, ulcerating enteritis induced by Mycobacterium avium subsp. paratuberculosis (MAP). It affects domesticated and wild ruminants throughout the world and causes significant economic losses. The opinion that the eradication of tuberculosis has resulted in the elimination of MAP-infected animals and that the disease no longer occurs in Poland, together with difficulties in the diagnosis of the disease, often complicated by other afflictions, the long period of its development and its dubious clinical picture, cause that herd infection might remain unrecognized for a number of years. Dyspepsia proceeding with periodical watery diarrhea was observed on a farm with 250 dairy cows located in Żuławy Wiślane, in the north-eastern region of Poland. By the elimination of epizootic diseases, the conclusion was that the most probable cause of the disease is paratuberculosis. It was proved by the serological tests of cows with clinical symptoms and cows which had manifested symptoms earlier. All blood samples from cows with clinical symptoms and two samples from clinically healthy cows had positive results for paratuberculosis. The subsequent serological test showed that 8.6% of the herd population was infected by MAP. An autopsy performed instantly after anaesthetizing the animals with clinical symptoms showed changes typical for paratuberculosis. Regular examination, the isolation and elimination of infected animals, as well protection of new born calves should be the method used to restrain paratuberculosis in the herd. It could be useful to certify the herds and transport the animals only between farms free from paratuberculosis.
An ELISA with a iipoarabinomannan as an antigen, developed for diagnosis of bovine paratuber- culosis, has been adapted for use in goats, and compared with complement fixation test. Kappa value of 0.62 indicated good agreement between CFT and the adapted ELISA and proved that the investigated ELISA may be helpful in diagnosis of Mycobacterium avium subsp. paratuberculosis infection in goats. The ELISA has been used to screen a randomly selected representative sample of Polish breeding goat population (21.78% of herds, 21.33% of goats). It has been demonstrated that only 2.42% of animals coming from 15.79% of herds were seropositive. Within-herd seroprevalence varied from 1.69% to 38.10%. Most of the infected animals (67.07%) were 3-4 years old. No seropositive cases were found in group up to 1 year old animals.
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