The exon distribution and primer position of koala TNFα (a), IL10 (b) and GAPDH (c) genes when in alignment with other marsupial and human sequences of the corresponding genes.

<p>The forward and reverse primer sequences used in the RT-PCR assays are highlighted in green and red, respectively. The PCR product has been designed such that it spans over an intron-exon junction.</p

B cell epitope mapping across four MOMPs in <i>Chlamydia</i> infected koalas after administration with placebo or vaccine.

<p>Response profile of a <i>C</i><i>. pecorum</i> F infected koala administered with a placebo (adjuvant only) across our MOMP peptide library (<b>A</b>). Response profiles of a naturally infected <i>C</i><i>. pecorum</i> G koala immunized with recombinant MOMP G (<b>B</b>), and a naturally infected <i>C</i><i>. pecorum</i> F koala immunized with recombinant MOMP G (<b>C</b>) across our MOMP peptide library. The blue line represents the absorbance of the pre-immunized sample. The red line represents the 20 weeks post-immunized sample. The green line indicates the background (Mean and two times standard deviation of negative controls).</p

TNFα and IL10 gene expression in koala PBMCs stimulated with UV inactivated <i>C. pecorum</i> in Group I, II and III animals.

<p>Samples were collected at 0, 12, 24 and 48 hours. Results are expressed as fold increased compared to GAPDH.</p

B cell epitope mapping across four MOMPs in naturally, <i>C</i><i>. pecorum</i> genotype G infected koalas.

<p>Response profiles of two wild koalas naturally infected with <i>C</i><i>. pecorum</i> G, Popeye (<b>A</b>) and Mars Bar (<b>B</b>) across MOMP peptide library and amino acid alignment of four MOMPs at VD4 epitope (<b>C</b>) (dots represent the consensus). Representative blot of plasma from the koalas infected with <i>C</i><i>. pecorum</i> G against four recombinant full length MOMP proteins (<b>D</b>). The blue line indicates the mean of absorbance of four <i>Chlamydia</i> free animals. The red line indicates the <i>Chlamydia</i> infected animal. The green line indicates the background (Mean and two times standard deviation of negative controls).</p

Amino acid sequence alignment of koala, brushtail possum and human TNFα sequences.

<p>(a) Koala TNFα sequence characterisation is done with reference to the human TNFα sequence. Presequence comprising of 76 amino acids and the secreted form of 156 amino acids is indicated by the horizontal red and green lines respectively. The hydrophobic transmembrane region and the intracytoplasmic region are marked out in red and green boxes respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059958#pone.0059958-Idriss1" target="_blank">[31]</a>. Asterisks indicate the receptor binding sites of the protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059958#pone.0059958-Eck1" target="_blank">[32]</a>. (b) Koala IL10 sequence characterisation is done with reference to the human IL10 sequence. Cysteine residues which form intramolecular disulphide bonds in human IL10 are marked in red boxes. Residues important for structural stabilisation are marked in green boxes. The six α-helices of the IL10 monomer are depicted by dotted blue lines <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059958#pone.0059958-Walter1" target="_blank">[33]</a>.</p

B cell epitope mapping across four MOMPs in naturally, <i>C</i><i>. pecorum</i> genotype F infected koalas.

<p>Response profiles of two wild koalas naturally infected with <i>C</i><i>. pecorum</i> F, Nixon (<b>A</b>) and Felix Pitt (<b>B</b>) across MOMP peptide library. Alignment of four MOMPs at an epitope at variable domain two (<b>C</b>), and two overlapping epitopes at variable domain four (<b>D</b>) (dots represent the consensus). The blue line indicates the mean of absorbance of four <i>Chlamydia</i> free animals. The red line indicates the <i>Chlamydia</i> infected animal. The green line indicates the background (Mean and two times standard deviation of negative controls).</p

Phylogenetic tree of (a) TNFα and (b) IL10 nucleotide sequence constructed using Geneious Pro 5.6.5 using Jukes Cantor, UPGMA tree build method with 1000 bootstrap repeat value.

<p>Phylogenetic tree of (a) TNFα and (b) IL10 nucleotide sequence constructed using Geneious Pro 5.6.5 using Jukes Cantor, UPGMA tree build method with 1000 bootstrap repeat value.</p

qrtPCR primers for TNFα, IL10 and GAPDH genes.

<p>qrtPCR primers for TNFα, IL10 and GAPDH genes.</p

Time course of licking responses in PND7 rats (males and females combined) in response to an injection of 0.3%, 0.4%, and 0.5% formalin into plantar surface of the left hindpaw.

<p>Data are presented as mean +/− SE. * <i>p</i><.05; **<i>p</i><.01; *** <i>p</i><.001, against other groups at the same time point.</p

Time course of flinching responses in PND82 rats (males and females combined) in response to an injection of 1.25%, 1.75%, and 2.25% formalin into the plantar surface of the right hindpaw.

<p>Data are presented as mean +/− SE. * <i>p</i><.05, against other groups at the same time point.</p