title: Composite testing for ante-mortem diagnosis of Johne's disease in farmed New Zealand deer: correlations between bacteriological culture, histopathology, serological reactivity and faecal shedding as determined by quantitative PCR.
abstract: BACKGROUND
  In the absence of overt clinical signs of Johne's Disease (JD), laboratory based tests have largely been limited to organism detection via faecal culture or PCR and serological tests for antibody reactivity. In this study we describe the application of quantitative faecal PCR for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in New Zealand farmed deer to quantify the bacterial load in cervine faecal samples as an adjunct to an existing serodiagnostic test (Paralisa™) tailored for JD diagnosis in deer. As ELISA has potential as a cheap, high throughput screening test for JD, an attempt was made to assess the sensitivity, specificity and positive/negative predictive (PPV/NPV) values of Paralisa™ for estimating levels of faecal shedding of MAP as a basis for JD management in deer.
  
  
  RESULTS
  Correlations were made between diagnostic tests (ELISA, qPCR, culture and histopathology) to establish the precision and predictive values of individual tests. The findings from this study suggest there is strong correlation between bacterial shedding, as determined by faecal qPCR, with both culture (r = 0.9325) and histopathological lesion severity scoring (r = 0.7345). Correlation between faecal shedding and ELISA reactivity in deer was weaker with values of r = 0.4325 and r = 0.4006 for Johnin and Protoplasmic antigens, respectively. At an ELISA Unit (EU) cutoff of >50 (Johnin antigen) the PPV of Paralisa™ for significant faecal shedding in deer (>104 organisms/g) was moderate (0.55) while the NPV was higher (0.89). At an EU cutoff of ≥ 150, the PPV for shedding >105 organisms/g rose to 0.88, with a corresponding NPV of 0.85.
  
  
  CONCLUSIONS
  The evidence available from this study suggests that Paralisa™ used at a cutoff of 50EU could be used to screen deer herds for MAP infection with sequential qPCR testing used to cull all Paralisa™ positive animals that exhibit significant MAP faecal shedding.
authors.affiliation: Disease Research Laboratory, Department of Microbiology & Immunology, University of Otago, Dunedin, New Zealand. rory.obrien@otago.ac.nz
authors.forename: Rory
authors.initials: R
authors.lastname: O'Brien
authors.forename: Alan
authors.initials: A
authors.lastname: Hughes
authors.forename: Simon
authors.initials: S
authors.lastname: Liggett
authors.forename: Frank
authors.initials: F
authors.lastname: Griffin
mesh_terms: D000822:Animal Husbandry / Q000379:methods; D000818:Animals; D058491:Bacterial Load / Q000662:veterinary; D057138:Bacterial Shedding; D003670:Deer / Q000382:microbiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005243:Feces / Q000382:microbiology; D016927:Mycobacterium avium subsp. paratuberculosis; D009520:New Zealand; D010283:Paratuberculosis / Q000175:diagnosis* / Q000382:microbiology / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D012720:Severity of Illness Index