Research Article: Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs

Date Published: November 16, 2015

Publisher: Public Library of Science

Author(s): Genevieve Housman, Joanna Malukiewicz, Vanner Boere, Adriana D. Grativol, Luiz Cezar M. Pereira, Ita de Oliveira e Silva, Carlos R. Ruiz-Miranda, Richard Truman, Anne C. Stone, Pamela L. C. Small.

Abstract: Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts.

Partial Text: Through the two-way transmission of pathogens between animals and humans, zoonotic diseases have a tremendous impact on modern human populations [1–2]. Mycobacterium is one important group of bacteria that has a long history of zoonosis [3]. In particular, the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium leprae respectively cause tuberculosis and leprosy—diseases that have afflicted humans for centuries and continue to contribute to major public health issues today [4]. Additionally, some mycobacterial pathogens can be transmitted among other animals, so it is important to identify the presence of these pathogens in potential animal reservoirs to assess the potential for disease spread and exposure threats.

In the New World, M. leprae naturally transfers between humans and armadillos, and this study takes advantage of this known zoonotic pathway to identify a useful method for surveying mycobacterial infection in wild primate populations. This paper both validated this single- and multi-copy loci qPCR method for the detection of mycobacterial pathogens in animal hosts using non-invasive cheek swab samples and further tested this technique in a case study to assess mycobacterial infections in wild marmosets.



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