New Zealand (NZ) is unique in that it has a population of more than 1.2 million farmed European red deer (Cervus elaphus), a farming practice not commonly employed internationally. Johne’s Disease (JD), caused by infection with Mycobacterium avium subsp. paratuberculosis (MAP) in deer presents as a unique syndrome and is increasingly recognised as a production limiting disease of concern to the NZ deer farming industry. In deer, JD manifests as an acute infection with progression from infection through to clinical disease or death occurring more rapidly than in cattle or sheep and with some particularly susceptible animals dying from the disease as early as eight months of age
. Consequently there is a need for accurate diagnostic tests for MAP infection in farmed deer, where JD may result in serious losses
Deer mount a vigorous immune response to MAP infection characterised by high titres of antibody
 and are capable of shedding large numbers of MAP organisms into the environment as the disease progresses from the paucibacillary to multibacillary state
. In contrast to cattle and sheep, young deer (<1 year of age) appear to be especially susceptible to challenge with the bovine strain of MAP
 although ovine strain MAP has also been implicated in cervine JD on occasion
. Nonetheless, most farmed deer herds that are affected by MAP do not suffer from overt clinical losses due to JD and good management practices appear to keep disease problems at a level that is acceptable within normal farming production systems in NZ. The motivation to introduce diagnostic methods that are appropriate and effective places extreme demands on diagnostic platforms used to support control of MAP in domesticated ruminants, for a disease that is only rarely obvious as a health hazard.
As is the case for all chronic, mycobacterial diseases in humans and animals, the development and validation of sensitive and specific diagnostic methods to diagnose infection and disease is particularly challenging. Perceived imprecision of available tests reflects the biology of mycobacterial infection and the chronology of the resultant immune responses triggered within the host, coupled with the existence of closely related and antigenically very similar mycobacterial species ubiquitous within the environment. These factors dictate that, however urgently they may be needed, the development of tests that more accurately and cost effectively diagnose MAP infection or JD in domestic animals is difficult. As no existing single test ticks every box in terms of sensitivity, specificity, turnaround and cost-effectiveness, combinations of different tests are necessary to achieve optimal diagnosis. Commonly utilised ante-mortem diagnostic tests for JD include immunodiagnostic tests for serum antibody by ELISA or organism based tests to detect the presence of the bacterium, such as faecal culture or PCR. The specificity of serological diagnostic tests may be compromised by common antigens shared by MAP, M. bovis and other saprophytic environmental mycobacteria that evoke an immune response in non-diseased animals. The sensitivity of serodiagnostic tests, particularly for subclinically infected animals in the early stages of JD, is also influenced by the dynamics of antibody production and the point at which a sample is assayed due to the predominantly cellular immune responses found in the early stages of disease, limiting the predictive value of the test
. Performance of commercially available serodiagnostic test kits is further challenged when considering host species other than the target species for which they were developed; commercial ELISA kits may have limited or no capacity to detect antibodies from different host species such as deer
. While faecal culture on Herrold’s egg yolk (HEY) medium has remained the definitive test for MAP infection this requires prolonged incubation periods of up to sixteen weeks and may be compromised by overgrowth by contaminating gut microflora
[9–13]. Radiometric tests (BACTEC™) have been developed as a substitute for traditional faecal culture tests and have been widely adopted as they speed up the time to detect mycobacterial growth
Diagnostic tests available to deer farmers in NZ have included conventional pooled and individual faecal culture on solid and BACTEC™ medium as well as indirect ELISA and agar gel immunodiffusion, each with associated strengths and weaknesses. In addition, there is available an IgG1 ELISA (Paralisa™) test developed specifically for the diagnosis of MAP infection in deer through the detection of antibody to denatured Johnin (PPD-J) and native Protoplasmic (PPA) antigens; the antibody responses to both test antigens are considered in parallel to arrive at a final result
[3, 15]. Initial studies in deer
[15, 16] have suggested that subclinically affected deer produce higher levels of seroreactivity (IgG1) than has been shown previously in cattle
 or sheep
. ELISA techniques such as the Paralisa™ lend themselves well to automation using laboratory robotics thereby reducing labour costs and turnaround time; this facet is one of the great strengths of ELISA as a screening technique as it allows whole herds or sub-populations of animals to be economically screened, quickly informing management decisions.
Because JD presents as an enteropathy and the intestine is the major site of colonisation of the causative agent, faecal samples are an obvious ante-mortem source material with which to attempt to diagnose MAP infection and faeces are considered to be one of the most important materials for diagnosis of JD in the live animal because it is possible to identify subclinical and clinical animals relative to the level of shedding of MAP organisms
. In deer, it has been reported that animals suffering from clinical JD are capable of excreting 5 × 106 colony forming units of bacteria per gram of faeces
 and that an infective dose can be as low as 103 organisms
. Internationally, attention has increasingly turned towards rapid, nucleic acid amplification based approaches for the confirmation and quantification of MAP bacilli in faecal samples
[11, 12, 22–28]. DNA amplification (PCR) based tests are attractive due to their low cost and high speed and have become established as a mainstream diagnostic methodology for a variety of pathogenic organisms
. The inclusion of fluorescent reporter dyes has allowed PCR to be used quantitatively to determine the bacterial load in individual samples allowing distinctions to be made between high, medium and low or non-shedders; this aspect of the technique could be an invaluable JD management tool for selective culling of infectious/shedding animals, to facilitate control in infected herds and accredit low-risk replacement animals for introduction into herds. A recent comparative study of qPCR
, solid and liquid culture and ELISA for detection of MAP in cattle based on 143 samples concluded using Bayesian methodology (independent of a gold standard) that test sensitivity for culture methods and qPCR, as well as test accuracy, are comparable. Sensitivity and specificity of qPCR in this case were reported to be 0.60 and 0.97, respectively; accuracy of qPCR (0.90) was comparable to both solid (0.91) and liquid (0.93) culture leading the authors to conclude that qPCR has considerable potential to quantify MAP in faecal samples.
While there are numerous reports describing quantitation of MAP shedding in cattle and sheep, there have been no reports describing quantitation of MAP shedding in cervine faeces by PCR or any observed correlation between faecal shedding and other diagnostic parameters in deer. The current study attempts to compare diagnostic precision of ELISA, qPCR, culture and histopathological assays for MAP infection and JD in farmed deer. An inhouse qPCR assay for faecal shedding was firstly validated using proficiency panels consisting of bovine faecal samples of previously determined MAP titre and subsequently applied to a panel of 663 cervine faecal samples for which matched blood samples were available.