Sensitive detection of Myobacterium avium subsp paratuberculosis in bovine semen by real-time PCR

Aims: To develop a fast and sensitive protocol for detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine semen and to make a critical evaluation of the analytical sensitivity. Methods and Results: Processed semen was spiked with known amounts of MAP. Semen from different bulls as well as semen of different dilutions was tested. The samples were treated with lysing agents and beadbeating and the DNA was extracted with phenol and chloroform. Real-time PCR with a fluorescent probe targeting the insertion element IS900 detected as few as 10 organisms per sample of 100 ¿l semen. PCR-inhibition was monitored by inclusion of an internal control. Pre-treatment with immunomagnetic separation was also evaluated, but was not shown to improve the overall sensitivity. Conclusions: Real-time PCR is a sensitive method for detection of MAP in bovine semen. Lysis by mechanical disruption followed by phenol and chloroform extraction efficiently isolated DNA and removed PCR-inhibitors. Significance and Impact of the Study: The high sensitivity of the applied method allows reliable testing of bovine semen used for artificial insemination to prevent the spread of Johne's disease, caused by MAP

Diagnosis and control of contagious caprine pleuropneumonia

Le diagnostic de la pleuropneumonie contagieuse caprine a souvent été jugé difficile, cette maladie pouvant être confondue avec d'autres mycoplasmoses des petits ruminants. Les symptômes et lésions peuvent être similaires et l'isolement de Mycoplasma capricolum subsp. capripneumoniae (MccF38) nécessite une bonne compétence technique. Une fois les souches MccF38 isolées, leur identification ne devrait pas poser de problème. De nouvelles techniques, telles que l'amplification en chaîne par polymerase, offrent désormais la possibilité d'identifier MccF38 directement à partir de prélèvements lyophilisés. Toutefois, l'isolement de souches MccF38 reste obligatoire pour une déclaration officielle d'infection. Jusqu'à présent, le test sérologique de référence était l'épreuve de fixation du complément, ses principaux inconvénients étant l'absence de sensibilité et de spécificité, ainsi que la brève persistance des anticorps décelés au moyen de cette technique. L'épreuve immuno-enzymatique (enzyme-linked immunosorbent assay : ELISA) de compétition, récemment mise au point, devrait désormais permettre de déterminer, à l'occasion de larges enquêtes sérologiques, la prévalence réelle de la maladie. Les traitements antibiotiques sont efficaces, mais ils ne peuvent prévenir la persistance d'un portage latent du mycoplasme. Unvaccin à mycoplasmes tués, adjuvé à la saponine, a été mis au poins au Kenya : il confère aux caprins une immunité d'environ un an. (Résumé d'auteur

16S rRNA based polymerase chain reaction compared with culture and serological methods for diagnosis of Mycoplasma pneumoniae infection.

Contains fulltext : 76527.pdf (publisher's version ) (Open Access)5 p

Interlaboratory comparison of real-time polymerase chain reaction methods to detect Coxiella burnetii, the causative agent of Q fever

The bacterium Coxiella burnetii, which has a wide host range, causes Q fever. Infection with C. burnetii can cause abortions, stillbirth, and the delivery of weak offspring in ruminants. Coxiella burnetii infection is zoonotic, and in human beings it can cause chronic, potentially fatal disease. Real-time polymerase chain reaction (PCR) is increasingly being used to detect the organism and to aid in diagnosis both in human and animal cases. Many different real-time PCR methods, which target different genes, have been described. To assess the comparability of the C. burnetii real-time PCR assays in use in different European laboratories, a panel of nucleic acid extracts was dispatched to 7 separate testing centers. The testing centers included laboratories from both human and animal health agencies. Each laboratory tested the samples using their in-house real-time PCR methods. The results of this comparison show that the most common target gene for real-time PCR assays is the IS1111 repeat element that is present in multiple copies in the C. burnetii genome. Many laboratories also use additional real-time PCR tests that target single-copy genes. The results of the current study demonstrate that the assays in use in the different laboratories are comparable, with general agreement of results for the panel of samples