ABSTRACT. Twelve killer whale (Orcinus orca) were hemmed in by ice floes, and nine died on the Aidomari coast in the Nemuro Strait in Rausu, Shiretoko, Hokkaido, Japan on 8 February 2005. Tissue samples collected from 8 whales were tested for Neospora caninum, Toxoplasma gondii, and Brucella species DNA by polymerase chain reaction (PCR) assay. Gamma-globulin isolated from blood samples by ammonium sulfate precipitation was tested for antibodies to these pathogens by means of agglutination tests and immunoblotting. None of the 8 tissue samples had antibodies to the pathogens, when subjected to agglutination tests. In immunoblotting, one sample (sample No.5) showed antibody binding to N. caninum antigens. In the PCR assay, none of the samples was positive. Further study is necessary to examine the prevalence of the pathogens in marine mammals inhabiting this area. Toxoplasma gondii and Neospora caninum are apicomplexan protozoa with a worldwide distribution that cause neuromuscular disease and abortion through transplacental transmission in warm blooded hosts. Brucella species, a facultative, gram-negative intracellular pathogen also cause abortion in mammals, as well as sterility. Ingestion in contaminated food is one of the infection routes of these pathogens. Recently, T. gondii infection has been reported in wild marine mammals, including several species of cetaceans [1,2,4,16,18,19]. As for Brucella species, infection has been observed in a wide range of species of marine mammals [6,17,22]. However, the source of these infections are still unclear and there has been no report of marine mammals being exposed to N. caninum in nature.On 8 February 2005, 12 killer whale (Orcinus orca) were hemmed in by ice floes, and nine died on the Aidomari coast in the Nemuro Strait, Rausu, Shiretoko, Hokkaido, Japan (44° 1.1'N, 145° 11.1'E). The killer whales are predators, preying upon cold-blooded and warm-blooded animals. They are cautious and avoid shallow waters. They live in groups which show strong social cohesion.We considered that the killer whales had a risk of exposure to the pathogens mentioned above, if the marine mammals which they had preyed upon were infected with these pathogens. In this regard, while there have been a number of studies on toxoplasmosis and Brucella infection in seals [9,10,14,21] there have been few on the prevalence of the pathogens in killer whales.Since mass-stranding of killer whales seldom occurs on the coast of this area, this was a good opportunity to study them as a pod. To investigate their exposure to the pathogens, antibody titers were obtained for blood samples from them by means of agglutination tests, and immunoblotting using T. gondii and N. caninum antigens. Tissue samples were tested for the presence of specific DNA fragments of the pathogens by the polymerase chain reaction (PCR).Nine of the dead whales were available for study as they were beached. For 7 of them, necropsy samples and tissue samples were taken (No. 1, 2, 3, 4, 5, 6 and 9) on day 5 to 7 post mortem. The rem...

Recent reports of toxoplasmosis in marine mammals raise concern that cold-blooded marine animals are a potential source of Toxoplasma gondii infection. To examine the transmissibility of T. gondii to fish, we observed the development of T. gondii tachyzoites inoculated into oviduct epithelial cells of goldfish (Carassius auratus) microscopically in vitro. Further, the survival period of tachyzoites inoculated into goldfish muscle was bioassayed in mice and through PCR analysis. In cell cultures at 37 C, both RH and Beverley strains of T. gondii tachyzoites had penetrated into cells at 6 hr post inoculation, and were multiplying. In cell cultures at 33 C, many tachyzoites of both strains attached to the host cells, but no intracellular tachyzoites were observed at 24 hr post inoculation. In the T. gondii inoculated goldfish kept at 33 C, tachyzoite DNA was detected in the inoculated region on day 3, but not on day 7. When inoculated goldfish were kept at 37 C, live tachyzoites were seen at the inoculation site on day 3, but not on day 7. These results suggest that T. gondii does not persist in fish.

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