Hammondia heydorni: evidence of genetic diversity among isolates from dogs

Canine isolates of Hammondia heydorni from Argentina, Brazil, and the United States were analysed for genetic diversity. A total of 14 isolates were tested for their ability to produce amplification using three PCR assays, one targeting the common toxoplasmatiid ITS-1 region and 2 amplifying novel, H. heydorni-specific loci, HhAP7 and HhAP10. While the ITS-1 fragments could be amplified from all isolates, only six isolates were capable of amplifying the fragments from the novel loci. The PCR products were further investigated for genetic diversity using restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) techniques. Polymorphism in the digestion pattern was evident only at the HhAP10 locus, differentiating two of the Argentinean isolates from the remainder. Mobility shifts on SSCP gels revealed that the two Argentinean isolates were not only different from the other four isolates, but also differed from each other, both at the HhAP7 and HhAP10 loci. The ITS-1 fragments of all isolates were identical by RFLP. However, two distinct mobility patterns resulted when the products were electrophoresed on SSCP gels. Based on the sequence data from the ITS-1 and the two random loci, the isolates could be broadly classified into two distinct groups, within which minor polymorphisms were evident. In contrast, very little heterogeneity occurred in the sequences of corresponding ITS-1 regions of Neospora caninum and Toxoplasma gondii isolates. Thus, it is concluded that there is a considerable degree of microheterogeneity among isolates of H. heydorni. This diversity should be taken into consideration while attempting to elucidate the systematics, diagnostics, and biology of H. heydorni in relation to N. caninum.