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

Abstract

Background: Mycobacterium avium subspecies paratuberculosis (MAP), the cause of Johne disease, is a slow growing mycobacterium. Viable MAP detection is difficult, inconstant and time-consuming. The purpose of this study was to compare a rapid phage/qPCR assay performed on peripheral blood mononuclear cells (PBMCs) with three standard methods of MAP detection: fecal MAP PCR; plasma antigen-specific IFN-γ & serum MAP ELISA hypothesizing that, if sensitive and specific, Johne animals would be positive and Control animals negative. We studied a well characterized herd of Holstein cattle that were naturally infected with MAP and their Controls.

Results: With phage/qPCR 72% (23/32) of Johne and 35% (6/17) of Controls were MAP positive. With fecal PCR 75% (24/32) of Johne and 0% (0/17) of Controls were MAP positive. With plasma antigen-specific IFN-γ 69% (22/32) of Johne and 12% (2/17) of Controls were MAP positive. With serum MAP ELISA, 31% (10/32) of Johne and 0% (0/17) of Controls were MAP positive. When phage / qPCR and fecal PCR results were combined, 100% (32/32) Johne and 35% (6/17) of Control animals were MAP positive. Younger Control animals (1-3 years) had significantly fewer plaques (25 ± 17 SEM) than older Controls (4-12 years) (309 ± 134 p = 0.04). The same trend was not observed in the Johne animals (p = 0.19).

Conclusions: In contrast to our hypothesis, using the phage/qPCR assay we find that viable circulating MAP can rapidly be detected from the blood of animals infected with, as well as those in the Control group evidently colonized by MAP. 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.

Keywords: Crohn disease; Johne disease; Mycobacteriophage; Mycobacterium avium subspecies paratuberculosis (MAP); Peripheral blood mononuclear cells (PBMCs); Phage assay; Quantitative PCR.

Fig. 1

Photographs of representative plaques. a A negative control. b Show the appearance of a small number of plaques; in this case taken from 10% of the buffy coat of 8 ml of blood. c Shows how the plaque numbers are obtained. Using the Stuart Colony Counter, each time a mark is made on the bottom of the petri dish, as the dish is depressed, the count is automatically tabulated. d A plate where the plaque count is 688 plaques, determined using the Stuart colony counter as demonstrated in panel C. e A plate where the number of plaques are “Too Numerous To Count.” In this particular case the inoculum was from 10% of the buffy coat. f 25% of the buffy coat from the same blood sample as shown in “e” was inoculated. In this plate the plaques are so numerous that the entire plate has been “Cleared”. No discrete plaques at all are discernable. Compare this appearance with that of the hazy appearance of the Control plate (Panel “A”) which is the appearance of the M. smegmatis lawn. (see Materials and methods)

Fig. 2

A screen shot of a qPCR comparing two quenchers: TAMRA and MGB. Studied are serial dilution of MAP DNA isolated from pure culture of “Dominic” (ATCC 43545). Note the more pronounced amplification with MGB

Fig. 3

A comparison of Control animals that are MAP positive or negative by the phage/qPCR assay. There are significantly fewer plaques in the plates of the Phage/qPCR assay in the MAP negative controls (Mann–Whitney two tailed p = 0.0018; displayed as **)

Fig. 4

A comparison of number of plaques between Johne animals that are MAP positive or negative by the phage/qPCR assay. There is no difference between the number of plaques in the two groups (Mann–Whitney two-tailed p = 0.7038)

Fig. 5

When Control animals are stratified by ages 1–3 (53%; 9/17). and 4–12 (47%; 8/17) years, the younger Control animals have significantly fewer plaques (25 ± 16) than the older animals (308 ± 134: Mean ± SEM: p = 0.04; unpaired two tailed t-test.)

Fig. 6

When Johne animals are stratified by ages 1–3 (19%; 6/32) and 4–12 (81%; 26/32) years, the trend for the younger animals to have fewer plaques (70 ± 31) than the older animals (223 ± 53: Mean ± SEM) does not achieve statistical significance (p = 0.19; unpaired two tailed t-test)

Fig. 7

A Venn analysis of the Johne animals, comparing phage/qPCR with the three conventional methods. In combination with fecal PCR, 100% of the 32 Johne animals tested MAP positive, with 15 cows positive in both tests (p(kappa)= 0.02) and no cows testing negative by both tests (a). When combined with plasma IFN-γ, there were four cows negative by both tests (p(kappa)  = NS: b). When combined with ELISA there were six cows negative by both tests (p(kappa)  = NS: c)