Comparison of a mycobacterial phage assay to detect viable Mycobacterium avium subspecies paratuberculosis with standard diagnostic modalities in cattle with naturally infected Johne disease

Johne disease [1], a chronic inflammatory intestinal disease, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Johne disease causes considerable financial loss to the agricultural community [2, 3] and is present worldwide [4]. There is increasing concern that MAP may be zoonotic, contributing to inflammatory bowel disease in humans, particularly Crohn disease [5‐12]. Although controversial, other human diseases have been considered to be associated with MAP, either directly or consequent to molecular mimicry [13‐16].

Many mycobacteria including MAP, Mycobacterium leprae and Mycobacterium tuberculosis grow slowly. The “gold standard” for the detection of MAP is culture, but this is both time consuming and difficult [17]. In cattle, primary isolation requires a minimum of 8 weeks, and in humans with Crohn disease up to 18 months [18]. When detectable by culture, this results in tardiness in making reliable diagnosis. Although not indicative of viability, when present in abundance, MAP qPCR is the most effective method of detecting the presence of MAP DNA [19]. Accordingly, it is desirable to develop reliable and rapid methods to identify the presence of viable MAP.

Mycobacterial phage assays, that provide rapid results, require the presence of viable mycobacteria that contain a cell wall that permits the phage to attach and then enter the mycobacterium. The phage then subverts the host cell’s metabolic process, producing copies of itself. This ultimately results in lysis of the infected cell with release of progeny phages in addition to host DNA. A mycobacterial phage/qPCR assay that identifies and quantifies MAP in bovine blood, specifically in peripheral blood mononuclear cells (PBMCs), would be a powerful addition to expedite the diagnosis of viable MAP in infected eukaryotes. Such assays have been used to diagnose the presence of viable MAP in bovine milk and feces [20, 21] in naturally infected cattle with Johne disease [22] and a herd that had been experimentally inoculated with MAP [23] and most recently in humans with Crohn disease and some of their controls [24].

The purpose of this study was to compare results of standard MAP diagnostic modalities (MAP fecal PCR, serum MAP ELISA and plasma antigen-specific MAP IFN-γ,) with a mycobacterial phage amplification assay followed by qPCR [20, 23, 25‐27] that permits rapid (< 24 h) diagnosis of viable MAP. Animals are from a herd of naturally MAP infected Holstein dairy cattle and their (nominally) non-infected controls, housed by the USDA at their facility in Ames, Iowa USA. Early in this index, initially blinded, plaque/qPCR study there were intriguing and unexpected results. Positive MAP signal was observed in some Control animals and negative MAP in some Johne animals. Consequently, we converted to an open label study comparing Johne animals with Controls.

We compared the results of the phage/qPCR assay with standard MAP diagnostic modalities (MAP fecal PCR, serum MAP ELISA and plasma MAP IFN-γ). Our data show that when Johne animals were tested the phage/qPCR assay, plasma IFN-γ and fecal PCR are equivalent in terms of percentages of MAP positive samples detected. At the cut-off used for serum ELISA, the detection of prior MAP exposure is less reliable. However, our Venn analysis shows that the combination of phage/qPCR and fecal PCR for MAP identified every Johne animal.

In contrast, the combination of phage/qPCR and either plasma IFN-γ or ELISA (mis)diagnosed 4 and 6 animals as MAP negative, respectively. Thus, the most sensitive combination of tests are the antigen identifying, phage/qPCR combined with fecal PCR. During the course of this study several conventional tests for Johne disease, in Iowa, were elevated in the Control group. However, only a single animal (5c) was moved from the Control herd to the Johne herd. As this is an “Intent-to-Treat” analysis, the data for 5c continues, in this report, to be presented as a Control animal. The other immunological changes were either transient or did not meet the threshold of positivity routinely employed in Iowa infected with MAP at or soon after birth may not manifest any disease until more than a year old and may continue to have productive milk production until age eight. We hypothesize that as the Johne disease becomes more manifest in animals originally in the Control herd, that the normal values of plasma IFN-γ or ELISA will eventually rise above the threshold for a positive test. Accordingly, we posit that this phage/qPCR assay may enable animals to be identified earlier in the course of infection, which could result in more effective preventative interventions.

Our observations differ from prior studies, where Control animals were all MAP negative [23, 26]. In this study, 35% of the Control animals were phage/qPCR positive. This is an indication that despite meticulous efforts of USDA farm staff to separate the control animals from those infected and provide an uncontaminated environment, some Control animals have been exposed to viable MAP and become colonized and/or infected.

The significantly fewer number of plaques in younger Control animals, is an intriguing observation. It may be ascribed to one of several factors. Possibly indicating a more robust immune response in the younger animals. Alternatively, it may be an indication that the tardy rate of replication of MAP might not manifest until animals have lived long enough for the colonizing/infecting MAP to replicate sufficiently to be more easily detected.

Not all mycobacteriophages identified as “D29” act identically in this assay. One strain could not be satisfactorily killed by the viricide (FAS) we use. Considerable time effort and expense was required to determine that it was the D29 and not the FAS viricide we used that was responsible. To date we have not identified why one strain could not be completely killed. We conclude that any investigator attempting to replicate our data should ensure that any strain of D29 used can appropriately be killed by the viricide they employ.

Our observation that apparently healthy control animals have circulating viable MAP should not be surprising. A competent immune response may be able to successfully cope with pathogen exposure. In humans, the presence of viable mycobacteria does not necessarily indicate active disease. One quarter of humankind (~ 1.9 billion people) have latent tuberculosis [28]. However, less than 6% (10 million persons) have active tuberculosis [29]. The DNA of M. leprae is found in 5% (calculated to be ~ 70 million) of nasal swabs in the subcontinent of India [30]. However their number of clinical cases of leprosy is ~ 400,000 [31]. Likewise, the failure to identify MAP by qPCR in some animals with clinical Johne is evocative of the inability to identify M. leprae in the majority of cases of tuberculoid leprosy [32, 33].

Corroborative evidence that immune competence is critical in successfully combating mycobacterial exposure is seen with the deleterious effect of genetic defects. A Nramp1 genetic defect is associated with susceptibility to Johne disease in cattle [34, 35], sheep [36], as well as leprosy [37] and tuberculosis in humans [38, 39]. In cattle, the presence of a NOD-2 defect results in a > 3-fold increase in the percentage of cattle that have Johne disease, compared to wild type genome cattle in the same herd [40], (see [41] for review of genetic defects influencing Johne disease in cattle). Likewise, a NOD-2 defect is also associated with an increased susceptibility to lepromatous leprosy [42]. Thus, genetic defects may, in part, explain susceptibility to Johne disease, tuberculosis, leprosy and inflammatory bowel disease and account for the presence of undiagnosed (non-MAP) mycobacteria.

Contrary to our initial hypothesis, using our phage/qPCR assay, we find viable MAP in a proportion of both Johne (72%) as well as Control (35%) animals. In the Johne herd, our data show that phage/qPCR assay, plasma IFN-γ and fecal PCR all detect a similar percentage of positive animals, albeit not always the same animals. In order to detect all animals in the Johne disease group, the most sensitive combination is phage/qPCR with fecal PCR. Phage/qPCR is exquisitely sensitive in detecting the presence of viable MAP in circulating PBMCs. Viable MAP was detected in 35% of the control animals with phage/qPCR, therefore this test identified additional colonized animals or animals with early or sub-clinical infections compared to the standardized, non-antigen-detecting, serum and plasma IFN-γ MAP diagnostics. These data indicate that the presence of viable MAP in blood does not necessarily signify that an animal must of necessity be demonstrably ill or be MAP positive by standard diagnostic methods. This phage/qPCR assay rapidly identifies MAP that must be viable. It has the potential to become a powerful tool in studying the prevalence and penetrance of MAP in human and veterinary diseases.

This study was approved by the Research & Development Committee of the James J. Peters VAMC Bronx NY (0720-07-035, MIRB 01002) and Material Transfer Agreement 16432 from USDA Ames Iowa.