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. 2021 Jul 29:8:665697.
doi: 10.3389/fvets.2021.665697. eCollection 2021.

Development of a Method to Detect Mycobacterium paratuberculosis in the Blood of Farmed Deer Using Actiphage® Rapid

Anton Kubala  1   2 , Tania M Perehinec  1 , Catherine Evans  1 , Andrea Pirovano  2 , Benjamin M C Swift  3 , Catherine E D Rees  1   2
Affiliations

Development of a Method to Detect Mycobacterium paratuberculosis in the Blood of Farmed Deer Using Actiphage® Rapid

Anton Kubala et al. Front Vet Sci. .

Abstract

Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of Johne's disease, which is an economically and clinically relevant pathogen for commercial deer production. The purpose of this study was to develop a method that could be used to rapidly detect MAP infection in deer using the Actiphage Rapid blood test. This test has previously been used to detect MAP in cattle blood following the purification of buffy coat using Ficoll gradients, however this method is quite laborious and costly. The purpose of this study was to develop a simpler method of blood preparation that was also compatible with deer blood and the Actiphage test. Initially differential lysis of RBCs using Ammonium Chloride-Potassium (ACK) blood lysis buffer was compared with the Ficoll gradient centrifugation method using cattle blood samples for compatibility with the Actiphage reagents, and it was found that the simpler ACK method did not have an impact on the Actiphage test reagents, producing an equivalent sensitivity for detection of low levels of MAP. When the two methods were compared using clinical blood samples from farmed deer, the ACK lysis method resulted in a cleaner sample. When a blinded test of 132 animals from 4 different production groups was carried out, the majority of the positive test results were found to be from animals in just one group, with a small number identified in a second group. The test results were found to be reproducible when a small set of positive animals were tested again 1 month after their initial testing. Finally a set of negative animals which had been previously screened using an ELISA test, all animals gave a negative Actiphage result. This study shows that this improved sample preparation method and Actiphage blood testing can be used to test blood samples from deer, and the full diagnostic potential of the method can now be evaluated.

Keywords: Actiphage; Mycobacterium paratuberculosis; bacteriophage; deer; qPCR.

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Conflict of interest statement

CR and BS are founder members and shareholders of PBD Biotech Ltd., and AP is an employee of the company. AP and CE are UoN students sponsored by the company, but they can declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. TP is an employee of UoN and has no commercial or financial relationship with the company. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Comparison of Actiphage pPCR test results using Ficoll and ACK methods to purify white blood cells. The Cq-values for the paired blood samples are compared using a scatter plot. Vertical and horizontal dashed lines are set at the cut off values for the Ficoll and ACK samples, respectively. Samples falling within each quadrant are colour coded and cut off values are given next to each line. The position of individual samples (#) referred to in the text are indicated.
Figure 2
Figure 2
Distribution of Actiphage MAP test results in commercial deer herd production groups. Blood samples were taken from 132 female deer prior to the rutting season. The samples were supplied to the lab blinded to production group. After the test results were available, the samples were unblended and sorted by production group. Green bars indicate negative MAP test results; red bars indicate positive MAP test results.
See this image and copyright information in PMC

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