Due to the late introduction and establishment of JD in Ireland, there are relatively few publications on Paratuberculosis in this country. This study set out to evaluate a diagnostic strategy for MAP in selected Irish herds by comparing culture and molecular assays. This has relevance in terms of implementing future screening strategies for vets in Ireland and elsewhere. It is important to state that due to the logistical problems associated with obtaining samples, and the difficulty involved in enlisting willing farmers to participate, it was impossible to standardise the testing among herds in terms of obtaining equal samples from each herd. However, given this shortcoming, some important observations and conclusions can be made at the individual animal level and within each herd.
In general, we found a poor correlation between culture results from individual animals (7.9%) and the corresponding first round PCR results (36%). This may be due to the degree to which an animal was shedding the organism, the current disease status of the animal at the time of sampling, or the significant loss of viable cells during the harsh de-contamination step. Collectively however, it was interesting to note that all herds that tested positive by culture were also PCR positive (H1, H2, H3 and H4) whereas the other 3 herds were negative using both methods. This is significant in terms of how PCR may be used to manage and detect Johne's disease rapidly within a herd, i.e a quick and relatively cheap PCR test from a representative number of animals could provide an early indication of the disease status of the overall herd.
Two herds were sampled twice in this study (H1 and H2) and both produced interesting results. For example, in the herd H1, the percentage of MAP culture-positive results varied from 75% in 2006 to 30% in 2007 (although the sample size was not comparable). In terms of Herd H2, the difference between culture and PCR results in 2007 and the same herd one year later was striking (n = 44 and n = 0). This may be explained by the culling of infected animals and the possible introduction of "JD free" cattle in 2008, as seen previously by Richardson et al. (2009)[25
]. It may also be explained by the selection of an alternative cohort of animals by the vets during the second visit. Due to the sensitivities involved in dealing with farmers involved in this voluntary study, limited information was made available to us regarding the degree to which the herd had been re-populated during the intervening year.
There is a significant lack of comparable studies in Ireland, and although the purpose of this project was not to draw conclusions regarding the prevalence of MAP in Ireland, it is interesting to note that our % recovery is almost double that seen by a similar study in 2002 (O'Doherty et al. in 2002[26
]. Using a similar experimental strategy this group tested 221 animals from 16 herds suspected of paratuberculosis infection and found a prevalence rate of 4.1%.
As seen in other studies, the difference between the culture results in two dairy herds (H2, 13% and H3, 14%) and a beef herd (H4, 3%) was significant. The common practice in Ireland for dairy herd managers to feed pooled colostrums and milk to calves relatively increases the risk of transmission through contaminated milk which is a significant risk factor [27
In terms of PCR and the reliability of molecular testing, a complete correlation was observed between the results of the IS900 and ISMAP02 targets. This was surprising but not unusual as observed in a similar study [23
The nested ISMAP02 PCR detected 29 further positive samples increasing the sensitivity of the assay by 10%. However, among these additional samples, when re-examined, 8 from herd H4 were found negative by 2 other specific nested PCRs (IS900 and F57) based on independent triplicate results. The presence of these false positive test results may come from a single assay problem for H4, or may be representative of a larger reliability issue with nested PCR in this context. As well as the 8 putative false negatives, 10 of the remaining extra positives came from herds H2 (2008), H5 and H7 which were negative by both culture and first round PCR (Table ). This may of course represent an excellent added sensitivity afforded by nested PCR for these 10 samples, or it could be indicative of potential problems with this extra test. We feel that the need for increased sensitivity must therefore be balanced with the risk of producing false positives among the results.
The presence of environmental mycobacteria (Mycobacterium non-chromogenicum
and Mycobacterium terrae
) within certain herds is another important finding although how it impacts on an animal's susceptibility to MAP infection is hard to assess (if at all). These mycobacteria belong to the M. terrae complex and were originally classified as non-pathogenic saprophytes, but their pathogenic status is changing. They have been isolated from soil and are usually present in clinical samples as an environmental contaminant. Mycobacterium nonchromogenicum
has also been isolated in cattle infected by bovine tuberculosis [28
]. Interestingly, the 18 samples from which Mycobacterium non-chromogenicum
and Mycobacterium terrae
have been isolated were PCR negative for MAP DNA which indicates that these two insertion sequence IS900 and ISMAP02 are not present in these mycobacteria.
Infected animals shed MAP in milk but also in the environment where MAP persists and survives in the soil, water and sediment [29
]. To minimise exposure of the human population as described previously by Hermon Taylor (2010), there is a need to understand all the potential reservoirs and all the transmission routes for MAP. If this pathogen is proven to be zoonotic, the implication for the dairy industry worldwide would be enormous [30
] especially in countries like Ireland where exported milk and dairy products are significant for the national economy. It follows therefore that all countries with a paratuberculosis related problem should be working towards a rapid, standardised and reliable indicator of infection. Based on our experience, PCR should play a significant role in this.