Hyuganetsu disease was caused by ingestion of raw gray mullet, indicating an endoparasite-born rickettsios is.
Attempts were made to isolate causative rickettsia from the parasites of the fish. Metacercaria of Stellantchasmus falcatus, homogenized or untreated, were inoculated into mice via peritoneal or oral routes. The mice were autopsied after 14 days and 30 days, respectively, and thereafter blind passages were done every 14 days with spleen homogenate. Four strains of rickettsia-like organisms were isolated by the former procedure and 6 by the latter.
These rickettsia-like isolates were studied for its characteristics comparing with Rickettsia sennetsu. They shared common morphological, biological and antigenic properties on the one hand such as:
1. They were visualized by Macchiavello staining of lymph node cells of infected mice; coccoid and pleomorphic.
2. They were unfiltrable through Chamberland L3 filters.
3. They were sensitive to heat treatments (50°C, 10 minutes), formalin (0.1%, 3 hours), phenol (0.5%, 3 hours) and ether (10%, 30 minutes).
4. They were sensitive to antibiotics.
5. The isolates were positive for direct immunofluorescence test with labelled anti-Rickettsia sennetsu antibody or for indirect method with convalescent patient serum of “Hyuganetsu” disease and labelled anti-human-γ-globulin antibody, and Rickettsia sennetsu was also positive for indirect method using convalescent mice serums infected with the isolates and labelled anti-mouse-γ-globulin antibody.
However, the pathogenicity and some other characteristics distinguished them from Rickettsia sennetsu on the other:
1. They did not kill mice while Rickettsia sennetsu did in 14 days.
2. Autopsy of the infected mice revealed no viscous exudate in the peritoneal cavity.
3. Mice immunized with the isolates acquired only a little cross immunity against Rickettsia sennetsu infection.
The possibility that the metacercaria of Stellantchasmus falcatus may carry Rickettsia sennetsu as well as the rickettsia-like organism is not inconceivable in the light of the fact that Nanophyetus salmincola, the vecter of salmon disease complex in dogs, can be concurrently infected with Neorickettsia helminthoeca and Elokomin fluke fever agent.

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The pathogenic organism of the Hyuganetsu disease, regarded as infectious mononucleosis, was isolated in Japan. However, many doubts remain in that the pathogenic organism is the causative agent of real infectious mononucleosis. On the other hand, the Tsutsugamushi disease seems to be included in the Hyuganetsu disease. The present authors have made investigations concerning the cultivation of the Nakazaki strain, which was isolated as the causative agent of the Hyuganetsu disease, in yolk sacs and the cultivation in tissues of whole embryonic mice. These results are obtained as follows:
1) The Nakazaki strain was successfully cultivated in yolk sacs only up to the 4th passage. 2) Cultivation in tissues of whole embryonic mice was successfully conducted. The infectivity of tissues increased to approximately 100-1000 times after 15 days of cultivation and the cultivation in vitro was successfully continued during at least 30 days by two passages.
3) At this time, the addition of hyaluronidase in the materials for inoculation promoted the establishment of infection and the cultivation was accelerated by this addition. 4) The pathogenic organisms propagated in the cytoplasma of cultivated tissues were either coccoid or oval in shape, approximately 0.3-0.4μ in diameter, under the photomicroscope. There was no remarkable variation compared with that of prepared from the living body.

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Electron micrographs of dengue viruses of Hawaiian strain and Mochizuki strain, which were adapted to mouse brain and chick embryo have been taken using purified virus materials.
Purification methods employed were as follows: (I) Combination of partial purification of the virus adsorption to and elution from erythrocytes and ultracentrifuge, (2) Combination of protamine sulfate precipitation and ultracentrifuge. Studies by electron microscope of purified preparations from infected mouse brain and chick embryo showed that the infective particles of dengue virus were spherical, uniform and aggregate, average 18±2mμ in diameter, and they were considered to be the virus.
The smaller particles of about 15mμ in diameter seen in infected mouse brain seemed to be normal components because the similarsized particles were observed also in the normal mouse brain.

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