Tsunezo Shiota

Light microscopical study of Blastocystis spp. in monkeys and fowls

An investigation of a Blastocystis species obtained from several species of monkeys and fowls was conducted to clarify the morphology of the organism, using light microscopical techniques including Giemsa, Heidenhain iron hematoxylin, Trichrome stains, iodine mount and phase-contrast microscopy. A comparison was made with Blastocystis hominis from humans. Blastocystis spp. were found in 15 out of 26 monkeys and in all of 12 fowls (10 chickens and 2 ostriches) examined. The behaviour of the parasites in the bowels was also examined, using paraffin-embedded sections. Microscope examination of the lumen contents at death revealed the organism in the caecum of monkeys and fowls. The organisms from faeces, cultures and the lumen contents of the caecum of humans, monkeys and fowls were similar, except for variations in the size and contents of the central vacuoles, which occupied the centre of the organisms.

Disseminated infection of Pneumocystis carinii in a patient with the acquired immunodeficiency syndrome.

This report describes the histopathology of a disseminatedPneumocystis carinii infecton in a 24-year-old Japanese male haemophiliac diagnosed as having the acquired immunodeficiency syndrome. He developed respiratory symptoms, andPneumocystis carinii pneumonia was confirmed by transbronchial lung biopsy. On the 70th day of hospitalization the patient died. Autopsy findings revealedPneumocystis carinii not only in the lungs but also in the stomach, jejunum, ileum, colon, mesoappendix, abdominal lymph nodes, diaphragm, and thyroid gland.

Bromodeoxyuridine labeling studies on the proliferation of intestinal mucosal mast cells in normal and athymic rats

The proliferation of mucosal‐type mast cells (MMC) in rat small intestine was studied using a bromodeoxyuridine (BrdU)‐labeling method. After 24‐h cumulative injections of BrdU into adult SD rats, 3.5% of MMC were labeled, while only 0.3% and 0.1% of mast cells were labeled in back skin and ear respectively. From the results, it was concluded that MMC division occurred more than 10 times as frequently as the division of skin mast cells. Similar results were obtained in athymic adult rats (F344/N Jcl‐rnu) in which the number of MMC was similar to that in heterologous animals. Thus, thymic factor(s) or T cells may not have an important role in MMC division in normal states. When SD rats were infected with Nippostrongylus brasiliensis, vigorous proliferation of MMC was brought about 13 to 15 days after infection. At that period, 40% of MMC were labeled by a single injection of BrdU, and 85% of MMC were labeled by 9‐h cumulative injections of BrdU, with the result that most MMC rapidly proliferated in the intestinal mucosa during this period. Mitotic figures of MMC were sometimes observed. On the contrary, hyperplasia of MMC was not observed in athymic rats infected with nematodes. Therefore, MMC hyperplasia after nematode infection is dependent on thymic factor or T cells, and its mechanism is different from that of MMC division in normal states, in which thymic factor(s) or T cells are not essential.

Provocation experiment: Pneumocystis carinii in several kinds of animals.

Provocation experiments of P. carinii in several kinds of laboratory animals were carried out in an animal house where P. carinii of rat strain had been maintained for several years. The organism propagated in cortisone treated rats, rabbits and mice without difficulty as reported by some authors in the past. The organism also propagated in the lungs of rats which had been bred with protein free diet. The present paper first indicates that guinea pig is susceptible to P. carinii. On the other hand, it becomes evident by repeated experiments, that golden hamster seems to be non-susceptible to P. carinii of rat strain even if enough dose of corticosteroid hormones is given. The presence of non-susceptible animal to a certain strain of P carinii is quite interesting from an epidemiological point of view.

Pneumocystis carinii infection in corticosteroid-treated cats.

Corticosteroids were administered to produce Pneumocystis carinii infection in cats. Six of 10 cats, injected intramuscularly for 97-141 days with 2 mg/cat twice weekly of betamethasone sodium phosphate, developed a light infection with P. carinii. Six of 7 cats, injected intramuscularly for 11-168 days with 10-25 mg/cat weekly of prednisolone acetate, also developed a light infection with P. carinii. There was no significant difference in the infection rate between the sexes and ages of the cats. Using Giemsa staining and Gomoris methenamine silver nitrate stain, P. carinii organisms were indistinguishable morphologically from human and rat P. carinii. The cysts and trophozoites were usually present singly or in small groups, and they always were adhering to the periphery of alveoli. The inflammatory changes were inconspicuous except for the fact that alveolar macrophages often were seen. Corticosteroid-treated cats should be useful in the study of experimental P. carinii infection. This is the first reported case of experimentally induced P. carinii infection in cats.

Morphology, development and behavior of pneumocystis carinii observed by light-microscopy in nude mice

The present paper describes the morphology, development and behavior of Pneumocystis carinii, especially of the trophozoites in the alveoli of conventionally raised BALB/c nude mice, by using phase-contrast microscopy, paraffin sections and semiultrathin sections embedded in JB-4 plastic. Under phase-contrast microscopy, trophozoites were ameboid in external appearance and 2 to 8 micron in diameter. Usually they have one or more lucid spherical vacuoles and one less lucid nucleus in the cytoplasm. Maturation and independence of intracystic bodies were observed in the developing cysts. The intracystic bodies were polymorphic, i.e. spherical, ameboid or elongated. The paraffin sections using the double staining of P. carinii with Gomoris methenamine silver nitrate (GMS) and Giemsa, showed a small number of mature cysts containing intracystic bodies and a large number of trophozoites within the characteristic honeycombed material in the alveolar spaces. In order to investigate the morphology and parasitizing behavior of the trophozoites and cysts as well as the response of alveolar tissue in more detail, semiultrathin sections of 0.5-2 micron thickness were made from the materials embedded in JB-4 plastic and stained with Giemsa. In lightly infected alveoli, some trophozoites and cysts were found to be closely attached to the Type I alveolar epithelial cells. In heavily infected alveoli, almost all alveoli were filled with trophozoites, cysts, debris of the host cells, and occasionally phagocytic cells (macrophages and neutrophils) containing cysts or trophozoites were found. It was noted that the cysts were very few in number (1%) compared with the number of the trophozoites in nude mice in a 0.4 mm2 area of 2 micron thick lung sections. The host tissue of nude mice in this study was not as strongly affected by the organisms as that in cortisone-treated rats and P. carinii pneumonia patients. In the present study the morphology of P. carinii found in nude mice was not different from that found in rats and in man.

Prevalence of pneumocystis carinii in Wild Rodents in Japan

During the period from November 1980 to April 1984, 292 small wild rodents were captured in 6 localities in Japan. Eleven out of 142 Apodemus speciosus, 1 out of 11 A. argenteus, 4 out of 40 Microtus montebelli and 2 out of 3 Mus musculus were positive for Pneumocystis carinii. The intensity of infection was generally low and the infection rate did not show any significant difference between sexes of the host animals. The seasonal prevalence of infection in A. speciosus was not remarkable, but was somewhat higher in winter to spring than in summer to autumn. Also, the infection rate in A. speciosus was higher in relatively heavy body weights than in the lighter or the heaviest weight groups. This is the first report of P. carinii infection in wild rodents in Japan. We propose A. speciosus, A. argenteus and M. montebelli as the new natural hosts for P. carinii. No morphological difference was found between P. carinii in wild rodents, in rats and in man as far as the present light microscopic examination is concerned.

Further light microscopic studies on morphology and development of Pneumocystis carinii.

In order to add more advance in light microscopic investigation of P. carinii, phase contrast microscopy partly followed by wet giemsa stain and semiultrathin section of the lungs embedded in JB-4 plastic were studied. In phase contrast microscopy, small and large sized trophozoites of P. carinii were clearly recognized. Although movement of trophozoite was not found, rhythmic movement of intracystic bodies with filopodia was often seen in mature cyst. Those living organisms were then directly stained with Giemsa by infiltrating under the coverglass. Thus the organism could be investigated both in unstained and stained conditions. It is noticed with interest that 8 intracystic bodies seem to fill up the cavity of cyst when cell division is completed, then they liberate and become independent into spherical bodies, followed by banana-shaped or amoeboid forms with motility. An emphasis was done that semiultrathin section made from JB-4 plastic embedded lungs was quite useful for investigation of P. carinii infection. Several sizes of mononuclear thin-walked trophozoites, mature and immature cysts, and empty cysts were more clearly distinguished than any other light microscopical method ever reported.