Mycobacterium avium subsp. paratuberculosis (MAP) is a slow-growing bacterium that causes Johne’s disease, a wasting disease in ruminants and other animals. A common test for Johne’s disease is the serum antibody ELISA test which monitors the humoral immune response of the animal following MAP exposure. However there are well known limitations of this test, for example the sensitivity can be extremely low, especially during subclinical stages of infection [1]. It has been established that there can be a bacteraemic phase in paratuberculosis, which has been demonstrated by both PCR [2] and culture [3, 4]. However the PCR cannot differentiate between live and dead cells and the sensitivity of the PCR assay is limited by inhibitory substances in the blood and the likelihood of there being a low number of MAP cells present [5]. However, studies that have compared the detection of MAP in blood by PCR to ELISAs have found an association but poor correlations between the tests [5, 6]. The long periods of time required for culture of this organism (even using automated systems) means that to date very few studies of blood samples have been completed so that the incidence, intensity and duration of mycobacteraemia is not known in any animal species.

We have previously described the use of a rapid, bacteriophage-based method (phage amplification assay) coupled with PMMS to specifically detect and identify viable MAP cells in the blood of naturally infected animals [7]. The organism was detected in milk and serum ELISA positive animals, but not from a certified Johne’s disease free herd that was milk and serum ELISA negative. The PMMS-phage method was employed here as components present in the whole blood inhibited the phage assay to such as extent that the sensitivity of the assay was not useful [7]. Using PMMS it was possible the capture the MAP cells and suspend them in a medium suitable for the phage assay. The stages of natural infection are difficult to ascertain in naturally infected cattle and given the known limitations of the ELISA tests for diagnosis of early infection this was not surprising, but it means that more data is needed to understand the relationship between bacterial load during mycobacteraemia and the blood ELISA results. The aim of this investigation was to apply the PMMS-phage assay to determine whether MAP could be detected in the blood of experimentally exposed cattle, where their stage of infection could be defined. Previously we have demonstrated that the MAP cells are found within the PMBC fraction of blood [7], and we have defined this as bacteraemia even though the MAP cells are intracellular rather than free in the blood stream. It is also possible that the assay would also detect MAP cells free in the bloodstream but to date we have not formally tested this possibility. The presence of viable MAP in cattle blood has previously been demonstrated in serum ELISA positive, inconclusive and negative animals by the PMMS-phage assay in an uncontrolled environment [7], thus the significance of mycobacteraemia is unknown, when compared to tests that are insensitive when used on subclincally infected animals, such as the ELISA test. As it can be assumed that the presence of this organism in the blood of the animal indicates that it has crossed the gut and disseminated, the results would be used to determine if evidence of disseminated infection correlates with the recorded immune response of an animal in a controlled environment.

Cattle infected with MAP may not show clinical signs of disease until years after exposure. Subclinically infected animals may shed MAP into the environment in their faeces and MAP cells can also be present in their milk, but both milk and blood ELISA testing on subclinical animals is notoriously insensitive [10, 11]. It is not known whether all animals that give a positive ELISA test result will go on to develop clinical Johne’s disease. The results of this study show the first application of the phage assay on experimentally infected animals and its comparison between faecal shedding and blood ELISA status. The results have shown that animals with variable blood ELISA status can have detectable levels of viable MAP in their blood. More data are required to better understand the relationship between the host immune response following MAP-exposure and disease progression, however having a clearly defined experimentally controlled study such as this is the first step in determining the usefulness of the phage assay. The ability to be able to detect MAP cells in blood samples from animals and obtain results within days (rather than within months), may aid the understanding of disseminated infection with regards to immune responses during disease progression and development of clinical disease and when this moves beyond a localised gut infection to a systemic phase.

It is also important to consider the type of bacteraemia detected in this study. As demonstrated here and previously, MAP can be detected in the PBMC fraction of blood and not others [7], so it is assumed that the MAP cells detected are internalised within PBMCs. In our experiments to establish the internalisation model, free MAP cells did not co-purify with the PBMCs supporting this model. The possibility exists however that bacteraemic animals have free MAP cells present in their blood that were not detected by our methods.

There were a number of differences in experimental design compared to a previous study in which we found viable MAP cells in whole blood ranging from 3–39 pfu.ml⁻¹ [7]. The assay used in the present study was transferred from University of Nottingham laboratory, where the assay was initially established and optimised, to the University of Sydney. The availability of different equipment and the use of different reagents (particularly the peptide-coated beads) meant that some aspects of the method were not fully optimised before the trial was carried out. Furthermore, the animals in this study were castrated males and therefore were not exposed to the same reproductive and lactation stress as the milking dairy cattle used in the previous study [12]. These factors may explain the differences in the plaque number results obtained between the two studies.

The PMMS method requires expensive reagents, such as biotinylated peptides and paramagnetic beads and the use of PMMS introduced more variability into the results gained as tested with paired samples. Using the well-established Ficoll-Paque method to separate PBMCs from whole blood removes the need for PMMS and means that the phage assay can be carried out directly, reducing losses during sample processing. However this does reduce the selectivity of the method and therefore other mycobacteria potentially present in the blood may also be detected by the phage assay. This means that there is a greater reliance on the performance of the end-point PCR identification step to confirm the identity of the cell detected by the phage. Although limited by the inefficiency of the PMMS, the results showed that the assay was specific; despite plaques being detected in two of the unexposed animals, no MAP DNA was detected in these samples, which probably represents some phage break-through in the assay. This has been noted before and emphasises the importance of combining the phage assay with a subsequent PCR-identification step.

In the group of exposed cattle, exposure to MAP occurred over 4.5 years before blood samples were taken for testing and none of the cattle were showing clinical signs of Johne’s disease. Approximately one third of MAP-exposed cattle had viable MAP in their blood that was detectable by the PMMS-phage assay. Although two animals were shedding MAP in their faeces, no MAP was detected in the blood of these individuals. This was interesting as it would have been assumed, if the disease had progressed to the stage of detectable level of shedding, that the animals would be more likely to have viable MAP in their blood. However the results suggested that this was not necessarily the case. In subclinically infected animals especially, faecal shedding is intermittent. This study examined a single time-point and only small volumes of blood were tested. It is also important to note that bacteraemia is a transient event that can happen during any stage of infection, whether that is in the early subclinical stage of infection or later [13]. Thus, these findings may indicate that the presence of viable MAP cells in the blood may also be intermittent relative to when the animals are found to be shedding MAP in their faeces or may be a rare event or may be the different forms MAP are found in the blood may relate to different pathophysiological manifestations that may or may not be related to the lack of correlation between faecal shedding and dissemination into the blood stream. Thus testing additional aliquots from a single time point may assist in determining the true likelihood of detecting viable MAP in the blood.

This is the first study to show that viable MAP may be present in the blood of experimentally MAP-exposed cattle prior to the onset of clinical signs and does not appear to be correlated to other measures, including faecal shedding and serum antibody responses, which are routine diagnostics used for Johne’s. The ability to detect viable MAP in the blood of subclinically infected animals may lead to increased sensitivity of diagnosis in the early stages of infection, but this requires validation. Here we have demonstrated the proof of concept in a controlled study where variations such as exposure (time and dose etc.) and environmental factors are limited [14]. Having a tool like the phage assay allows new questions to be asked possibly in a longitudinal study testing the blood, milk and faeces of animals to enable a better understanding of paratuberculosis disease progression, determine when systemic infection occurs in animals, and how this influences the serum and milk ELISAs, and faecal culture or PCR results.

MAP, Mycobacterium avium subsp. paratuberculosis; MPN, most probable number; PMMS, peptide-mediated magnetic separation