These included a 465 bp fragment of ompA that comprises the highly variable VD III and IV regions which were previously targeted in a range of phylogenetic and fine-detailed epidemiological studies [11, 21] and a 726 bp highly polymorphic fragment of the tarP gene. Phylogenetic analysis Phylogenetic reconstructions were performed under

both distance and maximum-parsimony frameworks. Distance analyses were performed using the neighbour-joining algorithm and the Tamura-Nei model of molecular evolution as implemented in MEGA. Maximum parsimony analyses were conducted by using the tree-bisection and reconnection method of branch KU55933 cost swapping and the heuristic search algorithm of PAUP* version 4.0b. Relative support for individual nodes was Ilomastat order assessed by nonparametric bootstrapping, with 1000 replications of the data. The pairwise-deletion option was chosen to remove all sites containing missing data or alignment gaps from all distance estimations. Optimisation of the branch lengths was done by using the maximum-likelihood method (using Modeltest to define the

evolutionary parameters [45]), subject to the constraint that all sampled sequences were contemporary (i.e., molecular clock was enforced). All rooted trees were constructed with mid-point rooting to facilitate genotypic comparisons of the outer topologies. Genotypic analysis The ability of each of the shortlisted genes to define specific genotypes within the koala populations was assessed, based on the nucleotide dissimilarity of sequences. To facilitate

comparisons with previous research on koala C. pecorum infections, a similar genotyping approach was adopted where nucleotide dissimilarity > 1% (based on multiple sequence Belnacasan cell line alignments of all koala strains for each gene) results in a new genotype [7, 8, 46] Recombination Recombination Detection Program (RDP) was used to test aligned sequences for recombination. This package utilises six published methods found to be sensitive for the identification Proton pump inhibitor of recombination and to yield the fewest false-positive findings [19]. The six methods are: RDP [47], GENECONV [48], Bootscan [49], MaxChi [50], Chimaera [51], and SiScan [52]. Different tests are applied to aligned sequences by each method to detect potentially recombinant regions [19]. The null hypothesis is clonality, i.e., that the pattern of sequence variation among the aligned sequences shows no indication of recombination [19]. Recombination was deemed to occur in a locus if clonality was rejected by three or more tests at a significance level of P < 0.001 [19]. GenBank accession numbers of novel sequences All novel C. pecorum sequences characterised in this study were submitted to GenBank and are available according to accession numbers HQ457440 to HQ457545. Results PCR amplification and sequence analysis of 10 candidate molecular markers from the koala C.