Multiplex PCRs for the specific identification of marsupial and deer species from faecal samples as a basis for non-invasive epidemiological studies of parasites

Abstract

Background: The specific identification of animals through the analysis of faecal DNA is important in many areas of scientific endeavour, particularly in the field of parasitology. Methods: Here, we designed and assessed two multiplex PCR assays using genetic markers in a mitochondrial cytochrome b (cytb) gene region for the unequivocal identification and discrimination of animal species based on the specific amplification of DNA from faecal samples collected from water catchment areas in Victoria, Australia. One of these assays differentiates three marsupial species (eastern grey kangaroo, swamp wallaby and common wombat) and the other distinguishes three deer species (fallow, red and sambar deer). We tested these two assays using a total of 669 faecal samples, collected as part of an ongoing programme to monitor parasites and microorganisms in these animals. Results: These two PCR assays are entirely specific for these animal species and achieve analytical sensitivities of 0.1-1.0 picogram (pg). We tested 669 faecal samples and found that some previous inferences of species based on faecal morphology were erroneous. We were able to molecularly authenticate all of the 669 samples. Conclusions: We have established PCR assays that accurately distinguish the faecal samples of some of the prominent large mammalian herbivores found within a water catchment system in the state of Victoria, Australia. The multiplex assays for marsupials and deer produce amplicons that are easily differentiable based on their size on an agarose gel, and can be readily sequenced for definitive species authentication. Although established for marsupials and deer, the methodology used here can be applied to other host-parasite study systems to ensure data integrity.

Primer design for extremely damaged DNA specimens of Asian rhinoceros species

“…Notably, these detection limits of all the primer sets in the present study were lower than in other studies by around 2-5 orders of magnitude. The limitation sensitivity tests in the other studies were in the range 0.02 ng-0.1 pg (Ali et al, 2015;Suwannarat et al, 2017;Koehler et al, 2020), while the range in the present study was 1-0.01 fg. However, the sensitivity results were tested using high-quality synthesized templates, which would certainly differ from using real DNA samples.…”

Wildlife Is a Potential Source of Human Infections of Enterocytozoon bieneusi and Giardia duodenalis in Southeastern China

“…Using the approach above, we have explored the microbes from various animals including wild deer ( Zhang et al, 2018b ; Koehler et al, 2020 ), marsupials ( Zhang et al, 2018c ), domestic alpacas ( Koehler et al, 2018 ), cattle ( Zhang et al, 2018a ), goats and sheep ( Zhang et al, 2020 ), companion cats and dogs ( Zhang et al, 2019 ), and humans ( Zhang et al, 2018e ). We also established a new phylogenetic classification system of overall 600 E. bieneusi genotypes ( Zhang et al, 2021 ).…”

Marked genetic diversity within Blastocystis in Australian wildlife revealed using a next generation sequencing–phylogenetic approach

“…Melbourne's water catchments are inhabited by sambar, red and fallow deer, which were introduced to Australia in the late 1800s from parts of Europe and Asia (cf. Koehler et al, 2020 ). Although some studies have tested faecal samples from these three species of deer for Blastocystis , most animal were captive (e.g., in zoos or on farms) (see Supplementary file 5 for references).…”

“…1 ). Host species were identified based on the morphological ( Triggs, 2004 ) and/or molecular ( Koehler et al, 2020 ) identification of scats.…”