ABSTRACT Infection with the cat-borne parasite Toxoplasma gondii has been detected in numerous Australian marsupials and can lead to severe disease (toxoplasmosis) in some cases. The seroprevalence of Toxoplasma on Kangaroo Island, South Australia has been reported to be higher than the South Australian mainland in macropods, cats, and sheep, suggesting an increased risk of infection on this island. However, Toxoplasma seroprevalence in small- and medium-sized terrestrial mammals was almost zero on the island and did not differ from that on the mainland. We surveyed Toxoplasma seroprevalence in koala ( Phascolarctos cinereus ) populations on the island and on the mainland and assessed their risk of infection and their role in the life cycle of Toxoplasma . All screened koalas from the island (n = 94) and the mainland (n = 63) were seronegative. This represents the largest Toxoplasma seroprevalence survey in this species and provided sufficient evidence to confidently demonstrate freedom from parasite exposure in both island and mainland populations at the time of the survey. Because koalas are extensively arboreal and predominately consume tree foliage, they appear to be at negligible risk of Toxoplasma infection. Furthermore, as koalas are rarely consumed by cats, we suggest that they have a minor role in the parasite's life cycle.

Toxoplasma gondii is a protozoal parasite with worldwide distribution that is able to infect a wide variety of mammals and birds. Our main goal was to screen for T. gondii antibody titers in a previously untested species, the spotted hyena ( Crocuta crocuta ); however, this goal first required us to investigate serological procedures that could be suitable for hyenas. Cats are the closest domestic relations of hyenas, so T. gondii antibody titers were first compared in 26 feral cats with specific or nonspecific fluorophore-labeled secondary reagents, i.e., anti-cat IgG or protein A. Substitution of anti-cat IgG with protein A caused a statistically significant drop in titer measurements in cats ( P = 0.01) with a reduction of the geometric mean titer equivalent to 1 doubling-dilution. The same procedures were then applied to captive spotted hyenas. Titers measured in 9 of 10 hyenas were identical whether anti-cat IgG or protein A was used as the secondary reagent: 5 had titers <1:16, 2 had titers of 1:16, and 2 had titers of 1:32. One hyena had maximum titers of 1:64 or 1:32 when anti-cat IgG or protein A was used, respectively. The use of protein A as the secondary reagent in serologic assays can be applied to a range of mammalian species and seems unlikely to affect test specificity; however, the use of protein A may reduce test sensitivity, as suggested in the present study using cats. Despite a control program, some exposure to T. gondii had occurred in the Zoo's spotted hyenas.

Oral administration of Toxoplasma gondii oocysts to cats (i.e., monoxenous transmission) typically induces patent infections in fewer than half of test subjects. In the present study, oral administration of T. gondii oocysts to 5 kittens induced a patent infection in 2 of them, but only 1 kitten shed enough oocysts to enable further study. Those monoxenously-produced oocysts were administered to another kitten, which produced a second generation of monoxenous oocysts, and then those were used to induce a third generation of monoxenous oocysts. These results provide a rationale to develop a strain of T. gondii that has efficient direct transmission. The isolate of T. gondii that was able to be passaged in this manner has been designated the Dubey strain and cultured tachyzoites have been donated to a repository.

During a canine distemper virus (CDV) outbreak in raccoons ( Procyon lotor ) from Cook County, Illinois, a juvenile female suffering from seizures was killed and necropsied. Gross and histologic findings of necrotizing encephalitis and proliferative bronchopneumonia were attributed to CDV infection and considered the cause of clinical signs. A section of cerebellum stained immunohistochemically for Neospora caninum revealed an approximately 40 μm diameter, round to oval cyst with a 2- to 3-μm-thick wall and filled with 1–2 μm diameter, round to oval bradyzoites. Polymerase chain reaction (PCR) results were positive for N. caninum using DNA extracted from the brain. Specific PCR for the closely related organisms Toxoplasma gondii and Hammondia heydorni yielded negative results. This case report provides histologic, immunohistochemical, and molecular evidence that raccoons are a naturally occurring intermediate host of N. caninum .

Neospora caninum infection is a common cause of bovine abortion. One method by which cattle can acquire infection is through ingestion of oocysts; however, this has not yet been proved to cause transplacental infection or abortion. In this study, 19 cows, pregnant between 70 and 176 days, were administered 1,500 to 115,000 oocysts through an esophageal tube. Seventeen of the cows became seropositive, indicating acquisition of infection, whereas 8 negative control cows remained seronegative ( P < 0.001). Offspring were examined using serology, histology, immunohistochemistry, parasite isolation, and polymerase chain reaction (PCR). Six offspring were infected and 1 of them was aborted. The aborted fetus had typical lesions and positive immunohistochemistry and PCR for N. caninum . All 6 cows with infected offspring had continuously rising antibody titers, whereas 10 of 11 infected cows with uninfected offspring had falling titers after an early apex. The risk of transplacental transmission was increased by later exposure times during gestation and by the dose of oocysts ( P < 0.01 for the 2 combined variables). The lowest dose of oocysts, when administered after the 160th day of gestation, caused transplacental infection in 1 of 2 animals. This study demonstrates that infection with N. caninum oocysts can cause transplacental transmission and abortion in cattle.

To determine whether deer can transmit Neospora caninum , brains of naturally infected white-tailed deer ( Odocoileus virginianus ) were fed to 4 dogs; 2 of these dogs shed oocysts. Oocysts from 1 of the dogs were tested by polymerase chain reaction and found to be positive for N. caninum and negative for Hammondia heydorni . The internal transcribed spacer 1 sequence of the new strain (designated NC-deer1) was identical to N. caninum from domestic animals, indicating that N. caninum is transmitted between wild and domestic animals, often enough to prevent divergent evolution of isolated populations of the parasite. NC-deer1 oocysts were administered to a calf that developed a high antibody titer, providing evidence that N. caninum from wildlife can infect cattle. In addition, N. caninum antibody seroprevalence was detected in 64/164 (39%) free-ranging gray wolves ( Canis lupus ), 12/113 (11%) coyotes ( Canis latrans ), 50/193 (26%) white-tailed deer, and 8/61 (13%) moose ( Alces alces ). These data are consistent with a sylvatic transmission cycle of N. caninum between cervids and canids. We speculate that hunting by humans favors the transmission of N. caninum from deer to canids, because deer carcasses are usually eviscerated in the field. Infection of canids in turn increases the risk of transmitting the parasite to domestic livestock.

Scarce information is available about Neospora caninum oocysts and sporozoites, in part because only small numbers of oocysts have typically been produced by experimentally infected dogs. We hypothesized that 1 reason for low experimental production of oocysts is that dogs have been fed tissues from experimentally infected mice instead of tissues from cattle (which are natural intermediate hosts of N. caninum ). In this study, 9 dogs were fed tissues from N. caninum –infected calves, and oocyst production was compared with 6 dogs that were fed infected mouse carcasses. The number of oocysts produced by dogs that ingested infected calf tissues (mean = 160,700) was significantly greater ( P = 0.03) than the number of oocysts shed by dogs that ingested infected mice (mean = 5,400). The second goal of our experiment was to demonstrate cyclical oral transmission of N. caninum between dogs and cattle. As few as 300 oocysts were used to successfully infect calves, and tissues from these calves induced patent infections in 2 of 3 dogs; oocysts from 1 of these dogs were administered to another calf, and tissues from this calf subsequently induced a third dog to shed oocysts. Oocysts were confirmed to be N. caninum using a species-specific polymerase chain reaction technique. In addition, sporulated oocysts were used to recover N. caninum in vitro after digestion in an acid–pepsin solution and inoculation of cell monolayers.

We previously reported that Neospora caninum can be induced to express BAG1, a bradyzoite antigen, within 3 days of culture under stress conditions. The main goals of the present experiment were to increase the expression of BAG1 in vitro (in part by extending cultures for 9 days), to observe parasitophorous vacuoles at various points of stage differentiation, and to test the ability of organisms produced in vitro to function like mature bradyzoites. Expression of BAG1 and of a tachyzoite antigen (NcSAG1) was monitored using a double-label immunofluorescence assay. For the purpose of this study, organisms expressing NcSAG1 were designated as tachyzoites, those expressing BAG1 were designated as bradyzoites, and those expressing both antigens were designated as intermediate zoites. The greatest percentage of intermediate zoites and bradyzoites (14%) occurred in bovine monocytes maintained for 9 days. These bradyzoites did not appear to be functionally mature; they did not induce patent infections in dogs, in contrast to bradyzoites that were produced in chronically infected mice. In vitro, large parasitophorous vacuoles contained either a pure population of tachyzoites or a mixture of tachyzoites and intermediate zoites, which is indicative of asynchronous stage conversion of organisms within a vacuole. Bradyzoites were first observed within small vacuoles on day 6, and bradyzoites never shared vacuoles with tachyzoites. This finding suggests that vacuoles containing bradyzoites may develop only if the cell is invaded by a zoite that has already begun bradyzoite differentiation. An alternative possibility is that cysts may develop if the establishing tachyzoite undergoes bradyzoite differentiation before multiplying. Cysts do not appear to arise from transformation of tachyzoites within large parasitophorous vacuoles.