Hammondia heydorni: Oocyst shedding by dogs fed in vitro generated tissue cysts, and evaluation of cross-immunity between H. heydorni and Neospora caninum in mice.

Hammondia heydorni is a coccidian parasite believed to be nonpathogenic for naturally-infected animals, but it is biologically and genetically related to Neospora caninum, a worldwide cause of abortion in cattle. The major aim of the present work was to determine whether dogs shed H. heydorni oocysts after consuming in vitro generated tissue cysts of the parasite. In addition, we investigated cross-immunity between H. heydorni and N. caninum in mice. Two dogs were fed cultured cells containing tissue cysts of H. heydorni mixed with canned dog food, and a third dog (negative control) received only non-infected cells mixed with canned food. The two dogs that consumed in vitro produced tissue cysts shed high numbers of oocysts, which were induced to sporulate and tested positive for H. heydorni by a species-specific PCR. The third uninfected dog did not shed H. heydorni oocysts in the feces. Oocysts shed by the dogs induced the formation of encysted bradyzoites of H. heydorni on KH-R cells. Nineteen BALB/c mice were employed in the cross-immunity study. Nine mice were orally inoculated with 1×105 sporulated oocysts of H. heydorni and challenged with N. caninum tachyzoites 30days after infection with H. heydorni. Other ten mice, which did not receive H. heydorni oocysts, were infected with 2×105N. caninum tachyzoites. Thirty days after challenging with N. caninum, all mice were euthanized and N. caninum DNA in their tissues was quantified by real time PCR. No statistically significant difference in N. caninum DNA concentrations were observed between the two groups. We concluded that in vitro generated cysts of H. heydorni are biologically active, because they induced oocyst shedding in dogs. As no cross-protection occurred in mice inoculated with H. heydorni and challenged with N. caninum, it is suspected that these parasites do not express significant numbers of homologous proteins during infection, or the immune response of BALB/c mice after H. heydorni infection was not sufficient.

In vitro cultivation of Hammondia heydorni: Generation of tachyzoites, stage conversion into bradyzoites, and evaluation of serologic cross-reaction with Neospora caninum.

Hammondia heydorni was in vitro isolated from oocysts shed by three dogs using a finite cell line from embryonal bovine heart (KH-R). The oocysts were purified and suspended in 2% potassium dichromate or 2% sulphuric acid for sporulation for 2-5 days at room temperature. The parasites were confirmed as H. heydorni by PCR using specific primers (JS4/JS5) and by negative reaction for Neospora caninum employing the primers Np6+/Np21+. H. heydorni sporulated oocysts (1 × 10(6)) from each dog were initially treated with sodium hypochlorite. For excystation of sporozoites, oocysts from one dog were lysed by ultrasound followed by incubation with 0.75% taurocholate. Excystation of sporozoites from the other two dogs was achieved by oocyst fragmentation with glass beads with no further chemical treatment. Tachyzoites were clearly seen in the cultures at three days post inoculation (dpi). Bradyzoite conversion and cyst formation were evaluated at different time points by using a polyclonal rabbit serum against a bradyzoite-specific antigen (anti-BAG1), and a rat monoclonal antibody (mAbCC2) against a cyst wall protein. Bradyzoites were firstly detected at 7 dpi. Between 18 and 21 dpi most of cultured parasites consisted of encysted bradyzoites. The H. heydorni cysts increased in size during cultivation and reached a length of up to 135 µm. The parasite was maintained in the bovine heart cells up to 4.5months. Sera from mice and sheep experimentally infected with H. heydorni oocysts reacted with H. heydorni by IFAT, but did not cross-react with N. caninum antigens using IFAT or immunoblot. These findings suggest that serological cross-reactivity between H. heydorni and N. caninum seems to be of minor importance.