Map infection of domestic-food-producing animals is associated with significant economic loss to the livestock industry worldwide. At present, preventive strategies to restrict the spread of Map in animal populations and to limit the economic loss are not satisfactory. This is because of the relatively low sensitivities of the currently available tests, which fail to detect many subclinically (first and second stage) Map-infected animals .
The present study describes an improved method for the detection of Map DNA in bovine faeces. Comparisons between the results of this new HYDEqPCR procedure and the other Map detection methods that are mainly used routinely by veterinary diagnostic laboratories (faecal culture, milk ELISA and milk qPCR) show that the proportion of Map shedders in an infected herd can be greatly underestimated. The use of this new HYDEqPCR procedure to efficiently and rapidly detect Map shedders will thus allow improved herd management for reduced Map transmission among animals.
To our knowledge, the detection limit of HYDEqPCR of 2.4 CFU/g Map-spiked faeces and/or 90 Map/g Map-spiked faeces are the lowest LOD values for Map that have been published to date. Considering Map-spiked faeces, three studies reported LOD values of 1,100 Map/g , 250 Map/g  and 3 CFU/g .
HYDEqPCR proved to be efficient in terms of DNA yield and purity, with dilution of the isolated DNA from the faecal samples not necessary, as our tests indicate that the sample matrix did not contain any inhibitory activities. With the use of the IAC in a qPCR assay, it is possible to distinguish between the false-negative and true-negative Map results. The mean efficiency of this DNA Map isolation procedure from faeces was 85 % (±21 %), which is better than has been previously reported . The primer set  and the probe for the IS900 qPCR were specifically selected to avoid IS900-like sequences that can be recognised in some other mycobacteria [15–17]. Despite the concerns over the specificity of this IS900 region, and hence possible false-positive results due to other IS900-like sequences, we selected it as the qPCR target. Indeed, many alternative targets (f57, HspX, genes 251 and 255, ISMav2, ISMpa1, ISMAP02) have been used for the detection of Map, and although f57, HspX and ISMAP02 are unique to Map, they are present in a single copy and six copies, respectively in the Map genome . Thus, IS900 remains one of the preferred target sequences for amplification of Map-specific loci, as its multi-copy presence (14–18 copies)  in the Map genome can provide for much more sensitive real-time PCR assays compared to assays searching for a single to six copies gene targets . In this case, many of the samples with low numbers of Map will still not be detected.
These limitations in sensitivity of the available diagnostic tests for the detection of Map infections at subclinical stages was one of the challenges that motivated the present study. Moreover, the studies that have reported that IS900-like sequences are the reason for false positive results have used conventional PCR assays [15–17], where it is known that the specificity is lower than that of real-time PCR assays. Furthermore, we took all of the necessary steps to avoid false-positive results in the present study. Thus, through our combination of the sequencing of the qPCR amplicons and all of our other qPCR specificity confirmation, we have been able to exclude false-positive results.
The results of HYDEqPCR were here compared with those of conventional faecal culture, milk qPCR, and commercial milk ELISA. The proportions of Map-positive samples from all of the animals identified with HYDEqPCR, faecal culture, milk qPCR and milk ELISA were 89 %, 19 %, 36 % and 1 %, respectively. This is in agreement with the results of a previously published study, where again, faecal PCR performed better than faecal culture and milk PCR . However, the data in the present study are contrary to a report that showed that more positive results were detected by milk ELISA, followed by faecal culture and then faecal PCR . However, a certain degree of discrepancy between studies must be expected, due to the differences in the protocols that are used across different laboratories. These differences can arise from the variable efficiencies of DNA extraction and purification procedures for the subsequent molecular testing, the media used, the preparation of the inoculum for culturing, and the various sensitivities and specificities of the molecular, ELISA and culture tests. Discrepancies between the results of different studies can also be affected by the selection of the animals tested, in terms of their ages and their Map infection stage, as well as of the overall herd prevalence.
Nevertheless, HYDEqPCR detected 70 % (98/141) positive samples in those that were deemed negative by bacterial culture. Based on these results of the bacterial cultures and the corresponding mean Cq values of HYDEqPCR, we can clearly distinguish between heavy and low-to-moderate shedders of Map. However, we cannot distinguish between low and moderate faecal shedders of Map. The lack of agreement between the low and moderate shedding as determined by faecal culture and the Cq values of HYDEqPCR casts doubt on these specific definitions of low and moderate shedding as determined by faecal culture. The proportion of low and moderate shedders as determined by faecal culture might indeed be underestimated, because a significant fraction of the Map can be killed during the decontamination procedure or because the clumping of several bacteria might form a single colony. Also, not all of the Map that are present in faeces can be cultured. On the other hand, the qPCR results can overestimate the viable Map concentration, as the non-viable bacteria are also determined with this method. Considering these positive and negative aspects of both of these diagnostic methods, there is now the argument that real-time PCR should become the gold standard instead of faecal culture, as over more recent years, various studies have shown better sensitivity and specificity for real-time PCR compared to bacterial culture .
In the present study, the high Cq values per PCR reaction across all of the age groups of the animals indicated low concentrations of Map in the faeces samples, which might also have been a reason for the relatively bad performance of the bacterial culture method. It has previously been reported that the sensitivity of bacterial culture often depends on the stage of the infection. The cows in the first stages of infection are known to shed low levels of Map in their faeces intermittently, and thus only 15 % to 20 % of these animals can be detected by faecal culture following a single test . The results of the highly sensitive HYDEqPCR suggest that the low-level Map shedders that were detected were either passive shedders (the so-called ‘pass-through’ of Map in the intestinal tract) due to oral consumption of Map or were actively infected (most likely in the first and second stages of infection). This is further confirmed by the good ‘body condition score’ of the animals and the absence of clinical symptoms that are characteristic of Johne’s disease. Moreover, a super shedder was identified within the tested herd. It is known that such super-shedder cows will not have clinical signs initially, and so they represent a major concern for the spread of Map to other cows. Previous studies in dairy herds have reported 40 % to 60 % as low shedders in herds . For many of the animals, this might reflect passive shedding after the consumption of water and feed that is contaminated through the presence of a small number of super shedders . Indeed, as little as 1 g manure from a super-shedder can result in passive shedding of Map in an uninfected cow [11, 24]. It has also been reported that low-level Map shedders are more likely to be truly infected with Map, than to just be undergoing passive shedding . However, whether cows are passive shedders or are truly infected cannot be differentiated ante-mortem. Despite this, the indication is that the incidence of subclinically manifested paratuberculosis is underestimated at present.
This high percentage of Map positivity in faeces was definitely unexpected, and especially in the group of calves aged ≤6 months. However, these results strongly suggest that the majority of these animals, including the calves, must have been in contact with Map, which might be explained in terms of bad hygiene management practices on the farm studied. Calves are most susceptible to Map infection, and on the particular farm where the present study was carried out, they were not separated from infected, shedding adult cattle. This contact between the calves and adult cow faeces has been described as the most important risk factor of Map transmission to calves . Calf to calf transmission  can also be one of the reasons explaining these high percentages of Map detection in the calf faecal samples. According to the results of hazards models on age at onset of faecal culture positivity based on results of individual culture for Map, the predicted proportion of cattle with an onset of shedding before 2 years of age is 30.3 % (95 % CI: 1.8 % - 91.8 %) in herds with an apparent prevalence ≥0.2 , and this is in agreement with the culture results of the present study. Moreover, in-utero infections cannot be excluded .
The milk qPCR here detected Map in 36 % (33/91) of the animals. The lower rate of positive samples detected by milk qPCR in comparison with HYDEqPCR were expected, as Map can be detected in milk only from animals in stages II-IV of Map infection . Milk qPCR is, therefore, inadequate to investigate the incidence of infection in a population of animals or in an individual animal. However, when the Map distribution in milk is investigated, milk qPCR remains a useful tool to provide information relating to Map circulation in the food chain. Milk sampling is also more convenient, as the samples can be obtained non-invasively. Here, milk qPCR defined four animals as Map positive that were Map negative by HYDEqPCR and faecal culture. This divergence can be explained, however, as it is known that the shedding of Map in faeces and milk is not synchronised .
With milk ELISA, we detect only 1 % (1/91) of the animals as positive for Map. This might be due to the low levels of the antibodies in these subclinically infected animals, such that they are not generally detected by commercial ELISAs . Indeed, the one animal that tested positive for this milk ELISA was recognised as a super shedder, as it had a very high concentration (3.2 × 106) of Map cells per g faeces when determined by HYDEqPCR. This concentration was also the highest concentration detected within the herd in the present study. Therefore, milk ELISA appears to be an inappropriate method for the detection of subclinically infected animals, at least when carried out with the kit that was used in the present study.
Agreements between HYDEqPCR and faecal culture, milk qPCR and milk ELISA were also searched for. There was some agreement between HYDEqPCR and faecal culture. Similar agreement between these two methods has been seen previously, according to the Cohen’s kappa coefficients . The low kappa coefficients in the present study for these two methods with relatively high specificities is an indicator of the higher sensitivity of HYDEqPCR when compared to faecal culture. The estimated agreements between HYDEqPCR and milk qPCR, and HYDEqPCR and milk ELISA were poor and slight, respectively, with no significance seen. Each of these methods detect different targets (bacterial DNA, antibodies), which have different disease dynamics and will thus be detected at different stages of a Map infection. The poor agreement between HYDEqPCR and milk qPCR might also be explained by the different timing of the shedding of Map in faeces and milk, as mentioned above.