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

Authors: GREENSTEIN, ROBERT - James J Peters Vamc; SU, L - James J Peters Vamc; GRANT, IRENE - Queens University - United Kingdom; FODDAI, ANTONIO - Queens University - United Kingdom; Turner, Amy; NAGATI, JASON - Columbia University; BROWN, SHELDON - James J Peters Vamc; Stabel, Judith

Submitted to: Gut Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/28/2021
Publication Date: 5/6/2021
Citation: Greenstein, R.J., Su, L., Grant, I.R., Foddai, A.C., Turner, A.M., Nagati, J.S., Brown, S.T., Stabel, J.R. 2021. Comparison of a mycobacterial phage assay to detect viable Mycobacterium avium subspecies paratuberculosis with standard diagnostic modalities in cattle with naturally infected Johne disease. Gut Pathogens. https://doi.org/10.1186/s13099-021-00425-5.
DOI: https://doi.org/10.1186/s13099-021-00425-5

Interpretive Summary: Johne's disease is a chronic, debilitating intestinal disorder in cattle characterized by diarrhea, reduced feed intake, weight loss and death. Cattle usually become infected as young calves by ingesting feces containing the causative bacteria. During the subclinical phase of infection the animal may be shedding the organism in its feces without showing any clinical signs of disease. In addition infected cattle can shed the organism into the milk, thereby presenting a potential mode of infection for humans. Infection with Mycobacterium avium subsp. paratuberculosis is difficult to diagnose and therefore to control. Development of accurate and sensitive diagnostic tests is critical for control of this disease. In the present study, the sensitivity and specificity of a phage/PCR assay for the detection of infection in white blood cells from infected cattle was evaluated. Results suggest that this assay would be useful for the detection of infection and detection would precede clinical signs of disease.

Technical Abstract: Mycobacterium avium subspecies paratuberculosis (MAP), the cause of Johne disease, is a slow growing mycobacterium. Viable MAP detection is difficult, in constant and time-consuming. The purpose of this study was to compare a rapid phage / qPCR assay performed on circulating white blood cells (PBMCs) with three standard methods of MAP detection. We studied a well characterized herd of Holstein cattle that were naturally infected with MAP and their Controls. Positives in the Johne animals were: Serum MAP ELISA plus/equal 31 percent; plasma antigen-specific IFN-gamma plus/equal 69 percent; Fecal PCR plus/equal 75 percent and phage / qPCR plus/equal 72 percent. In contrast, among the Control animals, the only positives were phage / qPCR (35 percent; 6/17) and IFN-gamma (12 percent; 2/17). MAP was detected in 100 percent (32/32) Johne cattle by combined phage / qPCR and fecal PCR. In contrast, in the Johne cattle, MAP was not detected with combined IFN-gamma and phage /qPCR in 12 percent (4/32) and with serum ELISA combined with phage /qPCR, MAP was not diagnosed in 19 percent (6/32). Younger Control animals (1-3 years) had significantly fewer plaques (25 plus/negative 17 SEM) than older Controls (4-12 years) (309 plus/negative 134 p equal 0.04). The same trend was not observed in the Johne animals (p equal 0.19). We conclude that viable circulating MAP can be quickly detected from the blood of animals infected with, as well as colonized by, MAP using the phage / qPCR assay. These data indicate that the presence of viable MAP in blood does not necessarily indicate that an animal must of necessity be demonstrably ill or be positive by standard diagnostic methods.