Application of the Actiphage® Assay to Detect Viable Mycobacterium avium subsp. paratuberculosis Cells in Fresh Sheep and Goat Milk and Previously Frozen Milk and In-Line Milk Filters.

Monika Beinhauerova,Iva Slana

doi:10.3389/fvets.2021.752834

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) is a well-known causative agent of paratuberculosis, a chronic infectious granulomatous enteritis of ruminants contributing to significant economic losses worldwide. Current conventional diagnostic tools are far from being sufficient to manage and control this disease. Therefore, increased attention has been paid to alternative approaches including phage-based assays employing lytic bacteriophage D29 to detect MAP cells. The aim of the present study was to assess the applicability and efficiency of the recently developed phage-based kit termed Actiphage® combined with IS900 real-time PCR (qPCR) for rapid detection and quantification of viable MAP in milk samples. We demonstrated that Actiphage® in combination with IS900 qPCR allows for rapid and sensitive detection and identification of viable MAP in milk samples with a limit of detection of 1 MAP per 50 ml milk. Using this method, the presence of viable MAP cells was successfully determined in 30.77% of fresh goat, sheep and cow milk samples originating from paratuberculosis-affected herds. We further used Actiphage assay to define the time-lapse aspect of testing naturally contaminated milk and milk filters frozen for various lengths of time by phage-based techniques. Viable MAP was detected in 13.04% of frozen milk samples and 28.57% of frozen milk filters using Actiphage-qPCR. The results suggest the ability to detect viable MAP in these samples following freezing for more than 1 year. The obtained results support the views of the beneficial role of this technology in the control or monitoring of paratuberculosis.

Highlights

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis manifesting after a long latent period in domestic and wild ruminants, e.g., cattle, sheep, goats and deer
This study focused on performance evaluation of the recently developed phage-based assay known as Actiphage combined with IS900 qPCR for rapid detection and quantification of viable MAP cells in milk samples of various origins
Only blood investigations for the presence of viable mycobacteria using the Actiphage assay have been described, with the method being successfully applied for the detection of MAP bacteremia in experimentally infected cattle [17] and newborn calves [21]

Summary

Introduction

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (or Johne’s disease) manifesting after a long latent period in domestic and wild ruminants, e.g., cattle, sheep, goats and deer. Clinical symptoms include chronic weight loss and wasting, diarrhoea, reduced milk yield, and animal death from exhaustion [1]. Viable MAP Detection by Actiphage in Milk clinical symptoms represent only a small proportion of the infected animals within a herd. Paratuberculosis is widespread throughout the world and herd-level prevalence rates are increasing globally in foodproducing animals, causing significant economic losses annually [3, 4]. Efforts have been focused on the development of alternative diagnostic approaches such as the phage amplification assay (PA), incorporation of which in diagnostic strategies of paratuberculosis could contribute to more reliable animal infection status assessment and, to a reduction in the prevalence of MAP-infected individuals in herds [5]

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