Table 1.
List of species used in primer design alignment.
Fig 1.
PCR amplification using T. muris primers on tissue, egg and faecal DNA.
T. muris primers amplified DNA from T. muris tissue DNA (Tm) and T. muris eggs (E) beaten for 5 and 10 minutes (numbers in superscript). Faecal DNA from T. muris infected mice when unbeaten (Ub) did not amplify, as did faecal DNA that was beaten (B) but carried out at the DNA extraction lysis temperature of 95°C (Numbers in black above lanes in °C). Bead-beaten faecal samples amplified when the extraction lysis temperature was dropped to 45°C. Arrow indicates the position of the expected 1,000 bp product. 1kb hyperladders were run (HL) and negative controls (X).
Fig 2.
Light microscopy of M. betsileo faecal smears.
Faecal smears from M. betsileo individuals were examined by light microscopy at x80 magnification. Nematode worm larvae (A) and adults (B) were observed. Bars are 100 μm.
Fig 3.
PCR amplification using Nem27 primers on faecal DNA from wild M. cowani amphibians.
DNA was successfully amplified using the Nem27 primers on bead-beaten M. cowani faecal DNA, regardless of whether 1 or 5 minutes of bead-beating were employed. However, amplification was better when 5 minutes of bead-beating were used (Δ indicates 5 minutes of bead-beating). Both M. cowani faecal DNA extracts from different individuals amplified (numbers in superscript). A 40 cycle thermocycling program was chosen due to the low DNA concentrations obtained by the extraction (4.3 ng/μl) and permitted amplification. Such results indicate that these amphibians have nematode stages in their faeces and may therefore be infected. Arrow indicates the expected 402 bp size product. A positive control (+) containing 1 μl of tissue extracted T. muris DNA and 4μl of faecal DNA was included. 1kb hyperladder was run (HL) and a negative control (X).
Fig 4.
PCR amplification using Nem27 primers on faecal DNA from ZSL London Zoo reptiles.
Nem27 primers successfully amplified both 5 μl and 10 μl (asterisked) of faecal DNA from S. crocodilurus (Sc), R. boulengeri (Rb), and T. g. whitei (Tw) indicating a likely nematode infection in these reptile species but not from Chamaeleo jacksoni (Cj) which exhibited no amplification. Arrows indicate the expected 400 bp size product. Positive controls (+) containing 1 μl of tissue extracted T. muris DNA and 4 μl of the relevant reptile faecal DNA were included, demonstrating an absence of PCR inhibitors in these extracts. 1kb hyperladders were run (HL) and negative controls (X).