First isolation of Hammondia heydorni from dogs in China

Fecal samples of 945 dogs were examined microscopically in 2 refuge facilities in China from March 2010 to November 2011. In 8 dogs, oocysts, 9-14 μm in size, were found. Their morphology was similar to those of Hammondia heydorni and Neospora caninum. Sporulated Hammondia/Neospora-like oocysts were fed to 2 dogs, 2 gerbils, 2 guinea pigs, and 2 KM mice; tissues from these inoculated animals were then fed to coccidia-free dogs to determine species susceptibility to these oocysts. Oocysts were not excreted in the feces of dogs or rodents inoculated with oocysts. However, the dogs fed the tissues of gerbils or guinea pigs that were inoculated orally with oocysts excreted fresh oocysts. Dogs fed tissues from guinea pigs inoculated with brain and muscular homogenate from guinea pigs that were fed sporulated Hammondia/Neospora-like oocysts did not excrete oocysts. These findings indicated that the oocysts from naturally infected dogs had an obligatory 2-host life cycle, with gerbils and guinea pigs as intermediate hosts. DNA isolated from these oocysts could not be amplified using N. caninum- and Toxoplasma gondii-specific primers. However, positive amplification with the H. heydorni-specific primers confirmed the presence of H. heydorni DNA in the samples. A comparison of the intron 1 sequence of the alpha tubulin gene with those from H. heydorni from dogs and H. triffittae from foxes showed that dog-derived oocysts possessed a different alpha tubulin gene. Both our dog-derived sequence and 2 previous alpha tubulin gene sequences from H. triffittae from foxes contained a 9-bp insertion relative to 3 sequences of H. heydorni from dogs. However, when the 9-bp insertion from H. triffittae sequences were compared, the 9-bp insertion in our dog-derived sequence had a nucleotide substitution. The present study, therefore, provides new evidence of genetic diversity among isolates from dogs. This is the first survey for H. heydorni in dogs from China.