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Rural Industries Research & Development Corporation
Diagnostic Tests for Johne’s Disease in Deer By Christopher Schroen, Tracey Bradley, Wendy McDonald and Robin Condron
September 2003
RIRDC Publication No 03/100 RIRDC Project No DAV-194A
JD is caused by an infection in regions of the gut by Mycobacterium avium subsp paratuberculosis (M. paratuberculosis), all domestic and many wild ruminant species are known to be susceptible.
Infection is usually established in young animals that will harbour the organism for many years.
The infection spreads within specific regions of the intestine and is eventually excreted in increasing numbers in faeces before the animal finally succumbs to clinical disease. Experience in New Zealand indicates that JD in deer herds may be seen as 2 possible syndromes, (a) sporadic cases in mixed age groups, and (b) outbreaks affecting up to 15% of young animals (Macintosh, 2002).
To allow livestock industries and animal health authorities to establish national programs for control of JD, effective diagnostic technology is required. Currently the only approved diagnostic test for JD in live deer is individual faecal culture (IFC). By contrast, strategies for pooled faecal culture (PFC) have been developed and approved for flock diagnosis in sheep and the ELISA is used extensively for cattle.
This project evaluated two tests for the diagnosis of JD in live deer. PFC and a modified ELISA were evaluated by comparison with IFC, agar gel immunodiffusion (AGID), culture of tissues (TC) and histopathology (HP). Sera for ELISA and AGID testing and faeces for culture were collected from 1222 deer from 7 properties on which JD had been diagnosed and 474 deer from 2 properties with no evidence of the disease. Of the 1705 animals sampled, 390 animals were sampled at slaughter where faeces and tissues were collected for culture and fixed tissues for HP. HP examination was performed on selected samples based on TC or IFC results The most sensitive method for confirming JD in deer was TC collected after slaughter. The HP examination was slightly less sensitive as it detected only 71% deer tested positive by TC. Among tests suitable for the diagnosis of JD in live animals, the IFC and ELISA detected 47% and 38-40% respectively of deer identified as infected by TC. These findings are consistent with previous reports of IFC and ELISA for the diagnosis of JD in cattle (Hope et. al., 2000).
The concentration of M. paratuberculosis excreted in faeces of infected deer demonstrated considerable variation. The numbers of the organism ranged from 101 to 106 colony forming units per gram of faeces (CFU/g) in faeces from deer with subclinical infection. Animals with clinical JD excreted on average 5x106 M. paratuberculosis in a gram of faeces. The detection limit (analytical sensitivity) of culture for IFC was 10-50 (CI 4-324) CFU/g. By comparison, culture of faecal samples for PFC 5 (one infected and four non-infected deer) was able to detect 200 (95%CI 30- 621) CFU/g and the detection limit for PFC 10 or 20 was approximately 104 CFU/gm of faeces.
The sensitivity of detecting an infected animal by PFC was reduced compared to IFC. The estimated sensitivity for PFC 5 compared to TC (estimated from IFC of 47% cf TC) was 32% for PFC 5 and 27% for PFC 10 and 20. Infected animals that were difficult to detect by PFC were animals with subclinical infection and less than 200 CFU/g of faeces. In a pooled faecal trial of a JD positive property where the prevalence of infection was 25%, PFC was positive for M.
paratuberculosis at PFC 5 and 20 (PFC 10 not performed). However, it is unlikely that PFC of more than 5 deer would be effective in detecting infection as a whole herd test where herd prevalence was less than 4% in a herd of 200 animals unless one or more animals were shedding above the detection limit.
An initial evaluation of the commercially available bovine ELISA kit (Parachek) demonstrated that the concentration of a conjugate used in this kit was found to be unsuitable for the diagnosis of JD in deer. The usefulness of more concentrated bovine conjugate, as well as Protein G and deer conjugates was investigated. Following optimisation experiments, each conjugate was further evaluated using sera from 172 deer with bacteriological and/or histopathological evidence of infection and in 210 IFC negative deer from two herds with no history of JD. The results were interpreted using three cut points: 0.1, 0.2 and 0.3 based on optical density (OD) readings at 450 nm. The use of the 0.2 cut point appeared to provide the optimal balance between sensitivity and specificity. The specificities and sensitivities obtained with the three different conjugates were similar. The sensitivities of the ELISA at 0.2 cut point were 38.1, 38.6 and 40.3% for the concentrated bovine conjugate, Protein G conjugate and deer conjugate, respectively. The specificities at this cut point were 99% for the concentrated bovine conjugate, 97.6% for the Protein G conjugate and 96.7% for the deer conjugate. The sensitivity of the AGID estimated against the results of TC was 20.5%. The specificity of the AGID using 72 sera from IFC negative deer was 100%.
For herd testing to provide market assurance for low risk of JD, diagnostic testing strategies are selected to detect infection in herds with 95% confidence of a prevalence of at least 2%. However the ability to detect disease at such a level of confidence and prevalence is dependent on the size of the herd and sensitivity of the test. Based on epidemiological statistics, results from this trial indicate that the IFC, ELISA, PFC of 5 and PFC 10-20 would identify JD at the 95% confidence in a herd of 200 deer with minimal prevalence of infection of 2.4%, 3%, 3.9% and 5%, respectively. It is unlikely that the AGID would be suitable for deer JD Market Assurance Program (MAP) as it is unlikely to identify animals not also identified by ELISA or faecal culture.
The findings of this study indicate that serological and bacteriological tests for JD in deer have similar performance to the tests used in cattle and sheep. Consequently it is recommended that these tests as modified for deer are appropriate to be adopted as standard diagnostic techniques and with consideration of the epidemiological limits with respect to the sensitivity of each test, are eligible for use in a deer industry MAP.
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