All positive secondary PCR products were analyzed using agarose gel electrophoresis and visualized with ethidium bromide staining. Products of the expected size were purified with a Biospin Gel Extraction Kit (BIOER, Hangzhou, China) and directly sequenced in both directions on an ABI 377 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA).
All nucleotide sequences obtained in this study were edited with each other and reference sequences downloaded from the Gen-Bank database using the BioEdit v 7.1 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html ), aligned using Clustal X 1.83 (http://www.clustal.org/ ).
Neighbor-joining trees of nucleotide sequences were constructed based on genetic distances calculated by the Tamura 3 parameter model implemented in the program Mega 6.06 (https://www.megasoftware.net/ ). Representative nucleotide sequences from this study were deposited in GenBank under accession numbers KX869922–KX869926 for E. bieneusi isolates in dogs and KX964627– KX964628 for E. bieneusi isolates in cats, respectively.
All nucleotide sequences obtained in this study were edited with each other and reference sequences downloaded from the Gen-Bank database using the BioEdit v 7.1 (
Neighbor-joining trees of nucleotide sequences were constructed based on genetic distances calculated by the Tamura 3 parameter model implemented in the program Mega 6.06 (