Mycobacterium avium subsp. paratuberculosis (Mptb) is the bacterium responsible for Johne's disease in ruminants, an enteric infection with a prolonged incubation period leading to granulomatous enterocolitis, weight loss and death. Mptb is not host specific and cross-species transmission occurs when there are high contact rates. As the organism is an obligate pathogen and parasite of animals, demonstrating its presence in clinical samples using microbial culture, microscopy with various staining methods and PCR are commonly used diagnostic tests in veterinary diagnostic and research laboratories. Furthermore, as there is active debate about the aetiological role of Mptb in Crohn's disease of humans, the culture of Mptb is also attempted in medical laboratories. However, culture of Mptb is not easy because the organism is extremely slow growing and cultures are prone to contamination with more rapidly growing organisms. In New South Wales, cultures are routinely incubated for up to 12 weeks before declaring a negative outcome (Whittington, 2009). An additional problem is the existence of fastidious strains of Mptb (mainly in sheep) which require specific culture media. In general Mptb can be divided into two broad taxonomic groups, S strains (derived from sheep) and C strains (derived from cattle and other species) with the former being notoriously difficult to grow. The S and C strains in reality represent the extremes of a taxon and a continuum exists, for example there is a group known as bison (B) strain and it too is difficult to isolate from clinical samples (Whittington et al., 2011).
It is obvious that a more rapid and simpler method than culture is required for detection of Mptb so research has been conducted in many countries for more than 15 years on DNA-based technology to detect Mptb. The most common relevant test is polymerase chain reaction (PCR), which may provide results within a day. Most PCR tests for Mptb are based on a gene called IS900 which exists in 15-20 copies in each Mptb genome. This gene is quite specific for Mptb, but related genes exist in obscure species of mycobacteria which normally live in the environment. These organisms sometimes find their way into faeces (presumably after ingestion with fodder) and may cause cross-reactions with IS900 (Cousins et al., 1999). For this reason, careful design and validation of PCR assays for Mptb is critical. PCR tests can be applied to faeces, tissues and other types of samples. Unfortunately some PCR tests for Mptb have been released commercially without validation or based on testing relatively small numbers of animals, and this has caused headaches for veterinarians who have had to try to manage disease control programs without adequate information about test performance (Alinovi et al., 2009; Orpin and Sibley, 2009). Nevertheless, PCR-based tests are already replacing culture of Mptb in routine testing for Mptb in some European countries.
In this report the results of validation of a new faecal PCR for detection of Mptb are described. This test is called the High Throughput Johnes (HT-J) test and will be offered through animal health diagnostic laboratories in Australia following approval by the Sub-Committee on Animal Health Laboratory Standards (SCAHLS) in 2013.
The HT-J test is a multi-step assay with the following stages (see Figure 1):
Extensive optimisation studies were conducted to determine the most suitable protocol for the test. The results of the HT-J test were compared with those of faecal culture to calculate the relative sensitivity and specificity of HT-J test. Faecal samples from sheep and cattle which were believed to have not been exposed to Mptb and from sheep and cattle from other flocks and herds which were known to have been exposed to Mptb were evaluated. To test the robustness of the new test, it was transferred to a second laboratory (Elizabeth Macarthur Agricultural Institute, EMAI) and the bovine samples were tested in both laboratories.
Bovine Johne's Disease.
Samples were sourced from more than 20 beef herds where a diagnosis of Mptb infection had been made in at least one animal. The herds were located in New South Wales, Tasmania and Victoria. Samples were collected from across the herds and were not biased in favour of older animals, clinical cases or ELISA reactors. Faecal samples were also obtained from unexposed properties from Queensland and Western Australia.
Approximately 1300 bovine faecal samples were tested at each laboratory. The apparent sensitivity of the PCR was 60-70% compared to faecal culture. However, approximately 3 times as many culture negative samples were detected in the faecal PCR test compared to faecal culture positive samples that were not detected in the PCR. Thus in the exposed herds that were tested there were a greater number of PCR positive samples compared to faecal culture positive samples (more than half as many again). The specificity of the test at both laboratories was >99%.
Ovine Johne's Disease.
Samples were sourced from more than 10 commercial sheep flocks in New South Wales with known exposure to Mptb. Other samples were from experimentally exposed sheep in research flocks at the University of Sydney. Due to the extreme difficulty of locating farms that had undergone extensive testing and had been proven to have had no exposure to Mptb, faecal samples were obtained from only one unexposed property. It was located in the New England region of NSW. Sheep from this property have consistently yielded negative results in tests for Johne's disease (faecal culture, serology) over more than a decade.
Approximately 600 faecal samples were tested. None of the samples from the unexposed flocks were positive in faecal culture. The specificity of the HT-J test relative to faecal culture was 99% and the sensitivity was more than 80%. About twice as many culture negative samples were detected in the PCR compared to faecal culture positive samples that were not detected in the PCR. Thus in the exposed flocks there were a greater number of PCR positive samples compared to faecal culture positive samples (about 10% more). Therefore the true sensitivity of the PCR was likely to be greater than faecal culture.
In a separate trial, the co-grazing of infected sheep with uninfected sheep at differing stocking rates was examined. Faecal samples collected from control sheep approximately 3 months after the trial commenced were tested. All samples at this timepoint were faecal culture negative. Infected donor sheep were culture and faecal PCR positive with high quantities of Mptb DNA in their faeces. Some otherwise unexposed sheep that were grazed in a paddock adjacent to a paddock with infected sheep had detectable Mptb DNA in their faeces.
During the validation of the HT-J test we examined samples that were representative of the range of samples which may be submitted for detection of Mptb. The study was not biased by inclusion only of faecal samples with high numbers of Mptb (which are relatively easy to detect). Overall the sensitivity of the PCR appeared to be greater than faecal culture because more animals from infected flocks and herds tested positive in faecal PCR than in faecal culture. However, the true infection status of the majority of these animals was not able to be determined in this project.
The results for both bovine and ovine Johne's disease suggest that the PCR test will detect a set of animals that overlaps with that detected by culture. A comparison of the faecal PCR test with faecal culture, tissue culture and histopathology is required to better understand the nature of positive results in the two faecal tests. It is already known that many animals with positive tissue culture results are negative in faecal culture because of stage of infection (Whittington and Sergeant, 2001). However we do not yet know whether it is these animals that test positive in the faecal PCR assay. An illustration of the conceptual performance of HT-J and faecal culture in an infected population is provided in Figure 2.
It is critical to be aware of several features that differ between faecal culture and faecal PCR:
PCR detects DNA, regardless of whether the organism from which it was extracted was alive or dead. Culture detects only living Mptb.
90 to 99% of the living Mptb in a faecal sample are destroyed during decontamination of the sample prior to culture. For this reason there must be >~200 live Mptb per gram of faeces to ensure successful culture. The losses of Mptb during preparation of a sample for PCR are probably less than the losses during culture.
There may be substances in clinical samples like faeces which inhibit PCR. These may vary from sample to sample, and may be affected by diet. The methods in the HT-J test minimise but do not eliminate the chances of inhibition.
For these reasons, culture results and PCR results for a given sample may differ, and so both tests work best at herd/flock level. The PCR, which appears to be more sensitive than culture, is especially appropriate as a herd/flock test because it may detect passive shedding of dead Mptb. On an endemically infected farm, there may be high levels of pasture contamination with Mptb, but over time most of the Mptb cells on pasture die (about 90% of them die each month). DNA persists intact within these dead cells for many months and possibly for years. These robust but dead cells can be ingested and pass through the gut, appearing in faeces. We suspect that generally a low signal in the HT-J test may be seen. However, individual animals which shed very large numbers of viable Mptb produce a very strong signal in the HT-J test. Thus the HT-J test is also useful for the detection of heavy shedding individuals. More experience in the application of the HT-J test in the field is required to fully understand its potential.
The new PCR test overcomes one of the greatest barriers to adoption of faecal tests for JD, namely the time delay. Including sample collection and transport to the laboratory, it is feasible to provide test results to a farmer within 1 week. The cost of the new test should be no greater than faecal culture. A further advantage is that the PCR test replaces culture for pooled faecal tests, and the same pooling rates should be applicable (5 samples for cattle, 50 samples for sheep).
The research described in this report was funded by Meat and Livestock Australia, and by Cattle Council of Australia, Sheepmeat Council of Australia and WoolProducers Australia through Animal Health Australia. Anna Waldron and Fran Galea provided skilled technical assistance during this study. Faecal samples were kindly submitted by many collaborators.