title: Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA.
abstract: BACKGROUND
  Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (Map) and it is one of the most important diseases in cattle worldwide. Several laboratory tests for Map detection are available; however, these are limited by inadequate sensitivity and specificity when used in subclinically infected populations. To identify Map shedders in subclinically infected cattle, we used a new, high-yield method for DNA-extraction from Map in faeces combined with quantitative real-time PCR (qPCR) for amplification of the insertion sequence IS900 of Map (HYDEqPCR). Evaluation of HYDEqPCR was carried out in comparison with faecal culture, milk qPCR, and milk enzyme-linked immunosorbent assay (ELISA), on 141 faecal and 91 milk samples, from 141 subclinically infected dairy cattle.
  
  
  RESULTS
  The qPCR proved to be highly sensitive, with a detection limit of 2 IS900 DNA copies/μl in 67 % of the reactions. It also showed 100 % specificity, as determined from 50 Map and non-Map strains, and by the sequencing of qPCR amplicons. The detection limit of HYDEqPCR was 90 Map/g Map-spiked faeces, which corresponds to 2.4 colony forming units/g Map-spiked faeces, with an estimated efficiency of 85 % (±21 %). When tested on the field samples, HYDEqPCR showed 89 % of the samples as positive for Map, whereas faecal culture, milk qPCR, and milk ELISA detected 19 %, 36 % and 1 %, respectively. Fisher's exact tests only show statistical significance (p ≤0.05) for the correlation between HYDEqPCR and faecal culture. The agreement between HYDEqPCR and milk qPCR and milk ELISA was poor, slight, and non-significant.
  
  
  CONCLUSIONS
  This study highlights the advantages of HYDEqPCR for detection of Map in subclinically infected populations, in comparison with faecal culture, milk qPCR and milk ELISA. HYDEqPCR can detect low-level Map shedders that go undetected using these other methods, which will thus underestimate the proportions of Map-shedders in herds. Identification of these shedding animals is extremely important for prevention of the spread of Map infection in an animal population. Due to the relatively high sensitivity and specificity of HYDEqPCR, it can be applied to test for Map at the herd or individual level, regardless of animal age or production stage. HYDEqPCR will allow early detection and control of Map in any population at risk.
authors.affiliation: Institute of Microbiology and Parasitology, Gerbičeva 60, Ljubljana, Slovenia. katarina.logar@vf.uni-lj.si
authors.forename: Katarina
authors.initials: K
authors.lastname: Logar
authors.forename: Rok
authors.initials: R
authors.lastname: Kopinč
authors.forename: Petra
authors.initials: P
authors.lastname: Bandelj
authors.forename: Jože
authors.initials: J
authors.lastname: Starič
authors.forename: Aleš
authors.initials: A
authors.lastname: Lapanje
authors.forename: Matjaž
authors.initials: M
authors.lastname: Ocepek
mesh_terms: D000375:Aging; D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary*; D005243:Feces / Q000382:microbiology*; D005260:Female; D008892:Milk / Q000382:microbiology*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*
chemical_list: D004269:DNA, Bacterial