DNA extraction and PCR analysis
DNA was purified from the samples using the QIAmp® DNA Mini Kit (Qiagen Pty Ltd) according to the manufacturer’s recommendations, then shipped to the UK on dry ice.
Purified DNA was subjected to an Anaplasmataceae family conventional PCR cPCR and a Babesia / Theileria spp. cPCR. The Anaplasmataceae family cPCR comprised a primer pair (EHR16SD GGTACCYACAGAAGAAGTCC & EHR16SR TAGCACTCATCGTTTACAGC) , which amplifies a 345 base pair (bp) fragment from the 16 S rRNA gene using the following thermal protocol: 95°C for 10 min and 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The Babesia/Theileria spp. cPCR comprised a primer pair (BabgenF GAAACTGCGAATGGCTCATTA & BabgenR CGGTAGGCCAATACCCTACCGTC) , which amplifies a 250–270 bp fragment from the 18 S rRNA gene using the following thermal protocol: 94°C for 10 min and 40 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 45 s. All reactions used 2x HotStarTaq Master Mix (Qiagen, Crawley, UK) with 200 nM each primer pair, 3.0 mM final MgCl2 and 2 μL DNA template in a total volume of 20 μL, and were performed in a Tetrad thermal cycler (MJ Research, Waltham, MA, USA). Positive and negative control reactions were used in each assay. Amplification products were visualised on an agarose gel following electrophoretic separation.
Purified DNA was also subjected to the following qPCR assays: species-specific qPCRs for each of M. haemocanis and ‘Ca. M. haematoparvum’, each duplexed with a GAPDH qPCR , pan-haemoplasma qPCRs , and an A. platys-specific qPCR (personnel communication: M Robinson, Acarus Laboratory, University of Bristol). Briefly, the canine species-specific qPCRs were performed using 2x HotStarTaq Master Mix with 200 nM species-specific haemoplasma 16 S rRNA gene primer pair (M. haemocanis: Mhf 1167 F GTGCTACAATGGCGAACACA & Mhf 1246R TCCTATCCGAACTGAGACGAA; ‘Ca. M. haematoparvum’: CMhp 124 F GGAGAATAGCAATCCGAAAGG & CMhp 252R GCATTTACCCCACCAACAAC), 100 nM haemoplasma TaqMan probe (M. haemocanis: Mhf 1188 T FAM-TGTGTTGCAAACCAGCGATGGT-BHQ1; ‘Ca. M. haematoparvum’: CMhp 192 T FAM-CTTCGGGAGCCCCGCGC-BHQ1), 25 nM canine GAPDH gene primer pair (17 F TCAACGGATTTGGCCGTATTGG & 106R TGAAGGGGTCATTGATGGCG), 50 nM canine GAPDH TaqMan probe (TXR-CAGGGCTGCTTTTAACTCTGGCAAAGTGGA-BHQ2), 4.5 mM final MgCl2 and 5 μl gDNA in a total volume of 25 μl. Briefly, the pan-haemoplasma qPCRs were performed using 2x HotStarTaq Master Mix with 200 nM haemoplasma 16 S rRNA gene primer pair (Haemofelis group: HF grp 567 F GGAGCGGTGGAATGTGTAG & HF grp 680R GGGGTATCTAATCCCATTTGC; Haemominutum group: HM grp 1061 F GGGGCCAAGTCAAGTCATC & HM grp 1199R GCGAATTGCAGCCTTTTATC), 100 nM haemoplasma TaqMan probe (Haemofelis group: HF grp 595P FAM-TYAAGAACACCAGAGGCGAAGGCG-BHQ1; Haemominutum group: HM grp 1096P FAM-TACCATTGTAGCACGTTYGCAGCCC-BHQ1), 4.5 mM final MgCl2 and 5 μl gDNA in a total volume of 25 μl. All haemoplasma qPCRs were performed in an Agilent MX3005P real-time PCR machine (Agilent Technologies UK Ltd., Wokingham, UK): 95°C for 15 min and 45 cycles of 95°C for 10 s and 60°C for 30 s, during which fluorescence data were collected. Briefly, the A. platys-specific qPCRs were performed using 2x HotStarTaq Master Mix (Qiagen, Crawley, UK) with 200 nM A. platys-specific citrate synthase gene (gltA) primer pair (APLgltA1.f AGGCGTGATTTCATCCTTCA & APLgltA1.r CACAGCAAGCTCTTCATTTCC), 100 nM gltA TaqMan probe (APLgltA1.p FAM-TGGCTGCGAAGTATCATGGGGA-BHQ1), 5.0 mM final MgCl2 and 5 μl gDNA in a total volume of 25 μl. All reactions were performed in an Opticon (Bio-Rad Labs. Ltd., Hemel Hempstead, UK) real-time PCR machine: 95°C for 15 min and 45 cycles of 95°C for 10 s and 64°C for 15 s, during which fluorescence data were collected. Positive and negative control reactions were used in each assay. Samples with discordant haemoplasma results (generic assay positive but M. haemocanis & ‘Ca. M. haematoparvum’ negative) were subjected to cPCR amplification using 2x HotStarTaq Master Mix, 200 nM of a universal Mycoplasma primer pair (HBT-F ATACGGCCCATATTCCTACG & HBT-R TGCTCCACCACTTGTTCA) , 3.75 mM final MgCl2 and 5 μL DNA template in a total volume of 25 μL, using the following thermal protocol: 95°C for 15 min and 50 cycles of 95°C for 10 s, 55°C for 15 s and 72°C for 30 s in a MJ Mini thermal cycler (Bio-Rad Labs. Ltd.).
Amplicons from positive cPCR results were purified using the NucleoSpin® Extract II Kit (Macherey-Nagel, ABgene, Epsom, UK), and subjected to DNA sequencing using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer. BLASTn analysis  was performed to compare the 16 S rRNA gene (Anaplasmataceae family & haemoplasmas) and 18 S rRNA gene (Babesia/Theileria spp.) sequences obtained to those in GenBank. A phylogenetic tree including existing haemoplasma species, as well as selected non-haemoplasma Mycoplasma species, was constructed using MacVector version 12 (MacVector, Inc., Cambridge, United Kingdom) for the 16 S rRNA gene using the neighbour-joining program from a distance matrix , corrected for nucleotide substitutions by the Kimura two-parameter model . The data set was re-sampled 1000 times to generate bootstrap percentages. The 16 S rRNA gene fragment of the novel haemoplasma species was deposited in the European Molecular Biology Laboratory Nucleotide Database (HE577612).