PCR products for both assays were separated by gel electrophoresis and visualised using a UV transmilluminator. Negative controls (dH2O) were included in each amplification round to control for PCR contamination. PCR products were purified with an Invitrogen PureLink™ PCR purification kit and sent to the Australian Genome Research Facility (AGRF) for sequencing using the Sanger dideoxy method [30]. Gene sequence names from each C. pecorum positive sample were derived from the population SCH 900776 mw from which the koala originated and the ID name assigned by the veterinarians (i.e. ‘Bre/Ned’ = Brendale population; animal name ‘Ned’). Sequence and

statistical analysis Alignments for each sequenced gene were produced using ClustalW Gefitinib supplier [31] and RevTrans [32] was used to reverse-translate all alignments. Non-coding genes were aligned based on their nucleotide sequence. The software package DnaSP 5.0 [33] was used to analyse the extent of sequence variation by calculating the number

of polymorphic and parsimony-informative sites, the average nucleotide diversity (p-distance) and Tajima’s test for neutrality (D-value). The Molecular Evolutionary Genetics Analysis (MEGA) [34] software package was used to calculate the number of synonymous and non-synonymous sites and subsequent dN/dS ratio using the Repotrectinib purchase Nei-Gojobori method [35]. The discrimination index (D.I.), based on Simpson’s index of diversity [36], was calculated to determine the differentiating and discriminatory capacity of each gene: where D = index of discrimination, N = number of strains in the sample, and n i = number of strains in group i. The index ranges from 0 to 1, with a value close to 0 indicating low genetic diversity and a value close to 1 indicating high genetic diversity [36]. Calculation of the D.I. requires at least three nucleotide sequences for analysis. Criteria for identifying genetic markers In order to select the most appropriate candidate

genes for further investigation, a shortlist of three genes, ORF663, incA and tarP (in addition to ompA), were selected based on their application in previous C. pecorum typing studies [21], in addition to several empirical criterions: Clomifene The average proportion of nucleotide distances (p-distance) should be ≥ 0.02 before intra-species differentiation may be attempted [37, 38], which can be calculated from an alignment containing two or more sequences [39, 40]. Furthermore, both highly constrained, slowly-changing molecular markers and highly variable genes under diversifying selection each have their advantages, disadvantages, and advocates [41], implying the importance of selecting genes under both positive and negative selection. Finally, the discrimination index (D.I.) for candidate markers should be > 0.