鶏伝染性喉頭気管炎の実験病理学的研究

摘要

The outbreak of Infectious laryngotracheitis (ILT) was observed by YOSHIMURA and ODAGIRI in Japan in 1962. In recent survey, this disease seems to become a major menace to the Japanese poultry industry. The present studies were undertaken to clarify the pathogenesis of ILT (chapter 1, 2), to establish the quick method for the diagnosis (chapter 3), to obtain the specific ILT lesions other than the respiratory organ in chicken (chapter 4), to clarify the pathology of ILT virus infection in chick embryo (chapter 5) and to clarify the susceptibility of Japanese quail to the virus (chapter 6). Experimental results are summarized as follows. Chapter 1 Immuno-histological findings in laryngotracheal lesions of the experimentally infected chicken Twenty-five chickens were inoculated with ILT virus, and laryngotracheal lesions were examined from day to day. Respiratory symptoms were observed from 3 days p.i., and some birds became fatal. From the 8th day, they began to recover. Macroscopically, severe inflammatory changes of laryngotrachea were seen from 3 to 7 days after exposure. Characteristic microscopic lesions of the disease, i.e., syncytial cell formation with intranuclear inclusion bodies were observed in the epithelial cells of mucous glands and adjacent epithelia at 2 days p.i., From 3 days p.i., these findings were also seen in the basal layer, and inflammatory edema accompanied with the infiltration of heterophils and macrophages were observed in the epithelial layer and lamina propria. These changes were most marked at 3 to 5 days p.i., From 7 days p.i., inclusion bodies disappeared. As the result of immunofluorescent studies, viral antigens were observed exclusively in the characteristic microscopic lesions of the laryngotrachea from 2 to 6 days p.i., and not in the propria and submucosa where infiltration of heterophils and macrophages was observed. At 6 days p.i., the viral antigens were abundant in the exudates of the laryngotracheal lumen as a result of epithelial desquamation, while fluorescence disappeared in the epithelial layer. Chapter 2 The ultrastructural findings in the tracheal epithlia of the experimentally infected chicken The earliest changes were observed in the nuclei at the 24 hours after infection. The nucleoplasm appeared homogenous due to accumulation of chromatin at the nuclear membrane. At 24 to 36 hours p.i., inclusion bodies of relatively high density were presented in nearly whole area of nuclei. Various types of immature particles were observed in association with the inclusion bodies. Virus particles which were formed in the nuclei were enveloped by two processes, i.e., nuclear budding and envelopment at the nuclear membrane, and at a segment of preexisting or newly-formed membrane in the cytoplasma. Loss of cilia and syncytial cell formation were detected in an advanced stage of degeneration on the second day after exposure. Cytoplasmic vacuoles were abundant and contained large number of enveloped virus particles. Enveloped particles of different sizes were found in the nuclear vesicles (ca. 110-160mμ) and in the cytoplasmic vaculoes (180-310mμ). Chapter 3 Quick smear method for the detection of inclusion bodies For the purpose of securing the rapid diagnosis of this disease, various smear methods for the detection of inclusion bodies were examined on the tracheal mucous membrane of chickens inoculated with the virus, and the following results were obtained. 1) GENDRE's fluid (alcoholic BOUIN), BOUIN's fluid and SCHAUDINN's sublimate alcohol were recommended for the fixation of smear. 2) Among many stains, the most excellent result was attained with hematoxylin and eosin staining, but GIEMSA's stain was not suitable for the purpose. 3) It was very difficult to discriminate virus DNA with acridin orange staining. Chapter 4 Extra-pulumonary lesions of the experimentally infected chicken For the examination of tissue susceptibility to ILT virus other than respiratory tract and conjunctiva, chickens were inoculated with the virus intravenously (group A), intracerebrally (group B), and by cloacal brushing (group C) and feather follicle brushing (group D), and they were examined successively. Respiratory symptoms were not observed in all of group. In group A, the liver showed multiple miliary necrotic foci form 3 days p.i., Characteristic microscopic lesions of the disease were observed in the hepatic cells and epithelial cells of interlobular bile duct and further in the epithelial cells of thymic medulla from 3 to 5 days. In group B, the liver was studded with miliary necrotic foci from 5 days p.i., Microscopically, characteristic lesions were observed in the epithelial cells of choroid plexus and arachnoidal methothelia from 1 to 5 days p.i., the hepatic cells and the epithelial cells of interlobular bile duct showed same lesions as those in group A at 5 to 6 days p.i., In group C, the mucous membrane of cloaca proctodeum and cloaca bursa showed reddening and catarrhal inflammation from 2 to 5 days pi., The characteristic microscopic lesions were observed in the epithelial cells of cloaca proctodeum from 1 to 4 days p.i., and also seen in the epithelial cells of bursal mucosa, as well as epithelial reticular cells of medulla of bursal follicle from 1 to 6 days p.i., In group D, feather follicle of the inoculation area became swollen as grain of millet or rice at 4 to 6 days p.i., Characteristic microscopic lesions were observed in the epithelial cells of feather follicle and feather root from 1 day p.i., They became severe, and were also found in the adjacent epidermal germinal epithelial cells at 3 to 5 days p.i., As the result of immunofluorescent antibodies studies in group A, C and D, viral antigens were confirmed agreeing with the appearance of intranuclear inclusions. Chapter 5 Pathology of ILT virus infection in the chick embryo ILT virus was inoculated respectively on the chorioallantoic membrane of 10-day-old chicken embryos (group A), into the amniotic cavity of 11-day-old embryos (group B), and into the yolk sac of 4-day-old embryos (group C). In group A, the liver was studded with multiple miliary necrotic foci at 6 to 9 days p.i., and hepatic cells of the foci showed microscopically characteristic lesions of ILT. ILT lesions were found further in the epithelial cells of bile ducts, oral mucosa, oral gland, pharyngeal mucosa, esophageal mucosa, gland of proventriculus, uriniferous tubules, thymic medulla, bursal mucosa and proctodeum. In group B, ILT lesions were found in the liver as in group A, and also in the epithelial cells of feather follicle, outer sheath and basal layer of feather, epidermal germinal layer, conjunctiva, nasal mucosa, infraorbital sinus, oral mucosa, oral gland, laryngotracheal mucosa, tertiary bronchi, air sac mucosa, esophageal mucosa, gland of proventriculus, pancreatic acini, intralobular duct of pancreas, bursal mucosa and proctodeum. In group C, characteristic histopathological lesions of ILT could not be found. Chapter 6 Susceptibility of the Japanese quail Japanese quails were inoculated with ILT virus by nasal instillation (group A), conjunctival (group B), laryngeal (group C), into air sac (group D), intracerebrally (group E), cloacal brushing (group F), and feather follicle brushing (group G). In group A, characteristic histopathological lesions of the disease were found in the epithelial cells of nasal mucosa and lacrimal duct in only one case. In group D, the lesions were observed in the epithelial cells of the primary, secondary and tertiary bronchi and air capillaries, but not in the larynx and trachea. In group E, the liver showed minute necrotic foci, which revealed focal necrotizing hepatitis, i.e., swelling, necrobiosis, presence of intranuclear inclusions, syncytial cell formation and necrosis of the hepatic cells, but no lesions were found in the brain and meninges. In group G, feather follicles of the inoculation area became swollen as grains of millet at 3 to 6 days p.i., Characteristic microscopic lesions were observed in the epithelial cells of feather follicles and feather roots from 1 day p.i., They became severe, and were also found in the adjacent epidermal germinal layer at 3 to 5 days p.i., As the result of immunofluorescent studies in group D, E and G, viral antigens were confirmed agreeing with the appearance of intranuclear inclusions. In group B, C and F, characteristic lesions of the disease could not be found.