魚類の細菌感染症の病理組織学的研究

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The author histopathologically studied Aeroｍonas hydrophila, Vibrio anguillicida and Edwardsiella tarda infections in Japanese eel (Anguilla japonica) and Streptococcus sp. and Nocardia kampachi infections in cultured yellowtail (Seriola quinqueradiata). Based on the histopathological findings, the author investigated characteristics of the infections and inflammation in fishes in the following pages. Chapter Ⅰ. Aeromonas hydrophila infection in Japanese eel 1) Fifteen naturally infected fish were collected from fish-culturing ponds in Shizuoka Prefecture March and October 1972 and in Mie Prefecture March 1975. The diseased fish externally showed red fins, and the red surface of the belly and ventral side of the tail. They anatomically manifested enteritis and often hemorrhage in the stomach. 2) The bacteria histologically multiplied in the mucous-like substance contained in the anterior part of the intestine. The region manifested mucous-desquamative catarrh accompanying lymphocytes infiltration into the epithelium and the tunica propria without an obvious, bacterial invasion. No metastatic lesion was formed but parenchymal alterations and circulatory disturbances occurred without a bacterial invasion. The alterations were characterized by the degeneration of hepatic cells, necrosis and the degeneration of epithelia of renal tubles, numerical atrophy of the renal hematopoietic tissue and necrosis of the splenic sheathed arteries. The circulatory disturbances were represented by hemorrhage in the liver, the stomach, the dermis, the gill epithelia and the renal glomeruli. The disturbances were also represented by exudation of serum and fibrin from altered glomerular capillaries. Based on the histopathological findings it was determined that the parenchymal alterations and the circulatory disturbances were caused by the toxic substances produced by A. hydrophila multiplying in the intestine. 3) Five strains of the isolated bacteria were intramuscularly injected at the rate of 0.5 and 1.0 mg per 100g to an individual fish. Three fish became moribund after 3 days but two fish survived for 10 days after the injection. In the moribund fish, the injected lesion was formed in the lateral musculature and manifested necrosis, edema, hemorrhage, stasis and angitis. A fatty change in hepatic cells, edema in the splenic sheathed arteries and nephrose occurred without an obvious, bacterial invasion. Chapter II. Vibrio anguillicida infection in Japanese eel 4) Twelve naturally infected fish were collected from fish-culturing ponds in Tokushima Prefecture summer 1975. The slight cases formed hemorrhagic patch-lesions on the body surface. The moribund cases formed hemorrhagic swollen-lesions on the body surface and anatomically manifested a congested liver, splenomegaly and enteritis. 5) The bacteria invaded the dermis, the subcutaneous adipose tissue and the red musculature in the patch-lesion, and moreover the bacteria spread and multiplied at the lateral musculature and the vascular wall of lateral-muscular veins in the swollen lesion. The infected lesions manifested necrosis, edema and hemorrhage. The moribund case underwent septicemia accompanying the generalized infection. The infectious spleen was necrotized to cause the blood sea. The infectious liver manifested marked sinusoidal congestion, hemorrhage and pressure atrophy of hepatic cells. In the intestine, the bacteria were disseminated at the tunica propria and the submucosa via capillaries and caused desquamative-catarrh enteritis. In the infectious kidney, the hematopoietic tissue was necrotized accompanying hemorrhage and bacterial emboli were often formed in the glomerular capillaries. The infectious gills manifested edema. 6) The isolated bacteria was intramuscularly injected at the rate of 0.2, 0.5 and 1.0 mg per 100g to nineteen Japanese eels. The fish became moribund in 18 to 24 hours after the injection with 0.5 and 1.0mg and in 3 to 4 days after the injection with 0.2 mg. They manifested the same pathological characteristics as the moribund cases of the naturally infected fish. 7) Based on the histopathological findings in the naturally and experimentally infected fish, it was determined that characteristics of V. anguillicida were strong invasiveness into the tissue and the cause of septicemia in the terminal case. 8) The sonicate of the bacteria was intramuscularly injected at the rate of 2.0 mg per 100g one to five times at intervals of 24 hours and 5.0mg per100g one to two times at the same interval. No fish died and no external change occurred during the experimental periods. The injected lesion was necrotized accompanying hemorrhage, infiltration of inflammatory cells and angitis. Hyperplasia of lymphocytes, lymphoblasts and splenocytes, infiltration of neutrophils and the formation of the germinal-center-like structure were caused in the spleen. Hyperplasia of immature neutrophils and activation of reticulo-endothelial cells lining sinusoids occurred in the renal hematopoietic tissue. Degeneration was caused in the hepatic cells. Based on the histopathological results, it was determined that the sonicate acted as the toxin and the antigen. Chapter III. Edwardsiella tarda infection in Japanese eel 9) Fifty-nine naturally infected fish were collected from fish-culturing ponds in Shizuoka Prefecture June to October and December 1972, February 1973 and in Mie Prefecture September 1975 and March 1976. The diseased fish were classified into two manifestation types according to the organ in which abscesses were initially formed ; the type of suppurative interstitial-nephritis (39 fish) and the type of suppurative hepatitis (20 fish). 10) In both types, the abscess consisted of immature neutrophils and the bacteria were phagocytized and multiplied in the neutrophils. Liquefaction of abscesses was followed by marked bacterial multiplication. The liquefied abscesses usually involved the blood vessels to form pus emboli and cause pyemia. The diseased fish of the type of suppurative interstitial-nephritis formed abscesses initially in the hematopoietic tissue of the kidney. They often produced suppurative lesions in the spleen, the stomach, the heart and the abdominal musculature in the pyemic condition. The terminal case underwent septicemia and manifested marked necrosis accompanying marked bacterial dissemination in the spleen, the liver and the gills. On the other hand, the diseased fish of the type of suppurative hepatitis formed abscesses initially in the liver. With growth of the abscesses, the renal hematopoietic tissue manifested marked hyperplasia of neutrophils and hematopoietic cells, and the infectious spleen manifested neutrophils infiltration and splenocytes proliferation. The septicemic case manifested necrosis accompanying marked bacterial dissemination in the spleen and the renal hematopoietic tissue. Based on the histopathological findings, it was determined that E. tarda was a pyogenic bacterium. 11) The isolated bacteria were intramuscularly injected at the rate of 0.5 and1.0mg per 100g to five Japanese eels. All fish formed a swollen lesion in the injected region of the body and became moribund after 2 days. Suppurative inflammation was caused in the injected lesion. No abscess was formed in the visceral organs. Marked necrosis was caused due to bacterial dissemination in the liver, the spleen and the renal hematopoietic tissue. Based on the results of the experimental infection, pyogenicity of E. tarda was proved and it was recognized that the experimental infection caused an injection disease far different from the natural infection. Chapter Ⅳ. Streptococcus sp. infection in yellowtail 12) Seventy-four naturally infected fish were collected from fish-culturing coasts in Kagoshima Prefecture autumn 1974 and 1975, winter 1976, in Nagasaki Prefecture autumn 1976 and in Mie Prefecture summer and autumn 1975, 1976 and 1977. 13) External characteristics were represented by necrotized eyes, exophthalmos, formation of white protuberances containing pus or caseous matter on the inner surface of opercles and production of swollen lesions containing pus or caseous matter in the caudal peduncle and basal regions of fins. The diseased fish manifested one of the above characteristics but often two of them. Anatomical changes were represented by peri-epicarditis and color changes or congestion in the liver. 14) In the eye lesion the bacteria penetrated and multiplied in the eye-lid, the conjunctiva, the cornea, the sclera, the adipose tissue surrounding the eye-ball and in the oculo-motive musculature. The infected lesion manifested necrosis, edema, fibrin deposition, thrombosis, hemorrhage and infiltration of neutrophils and macrophages in the acute case. The chronic case manifested granulomatous inflammation in the eye lesion. The protuberances in the opercles and the swollen lesions in the caudal peduncle and the basal regions of fins were due to the formation of abscesses in the subcutaneous tissue in the acute case. In the chronic case, they were due to the formation of granulomas enclosing the abscesses. Metastatic lesions were usually formed in the peri-epicardium due to bacterial dissemination into the pericardium. Fibrin deposition and macrophages infiltration occurred in the acute case. The disseminated bacteria were phagocytized and multiplied in the marcophages. In the chronic case granulomas were formed to enclose the bacteria and the inflammatory exudates on the peri-epicardium. The metastatic lesion was not obvious in the other visceral organs. Parenchymal degeneration and necrosis or congestion occurred in the liver. 15) Experimentl infections were implanted by an intramuscular injection with the isolated bacteria at the rate of 1.0mg per 100g, smearing the bacteria onto the eye at the rate of one loopful cells per one fish and the oral administration with the bacteria at the rate of 20mg per 100g. Five fish (200g body weight) were used in each experiment. The injected and smeared fish became moribund after 5 to 7 days and they manifested granulomatous inflammation in the infected and metastatic lesions.16) Based on the results of the naturally and experimentally infected fish, it was determined that Streptococcus sp. was a bacterium causing granulomatous inflammation. Chapter V. Nocardia kampachi infection in yellowtail 17) Seventeen naturally infected fish were collected from fish-culturing coasts in Mie Prefecture autumn and winter 1975 and 1977 and in Okinawa Prefecture autumn 1975. The diseased fish were classified into four manifestation types according to external and anatomical views. Type 1 was the abscess-type characteristic in the formation of the large abscesses in the subcutaneous adipose tissue and the spleen. Type 2 was the nodule-type characteristic in the formation of small, white, nodule-appearing lesions in the lateral musculature and viscera. Type 3 was the airbladder-nodule-type characteristic in the marked formation of nodule-appearing lesions especially in the airbladder. Type 4 was the branchitis-nodosa-type characteristic in the formation of multi-nodules in the gills. 18) Among the types 1,2 and 3, the infected lesions histopathologically manifested the same form of inflammatory responses ; at first, two different shapes of abscesses, then abscess-granulation type of lesions and granulomas terminally. The large abscess was characterized by marked infiltration of neutrophils and a tendency of being formed in the subcutaneous adipose tissue and the spleen. The small abscess which was observed as a nodule-appearing lesion with the naked eye, was characterized by the formation of a layer consisting of macrophages to wall off the pus and a tendency of being formed generally. In both types of abscesses the bacteria markedly multiplied extending branches and forming many druse. In the abscess-granulation type of lesion, the pus was walled off by a layer consisting of the granulation tissue and epithelioid cells. In the granuloma thick layers of epithelioid cells walled off the suppurative exudates, in which the bacteria decreased in number and were trasformed into short rods. In the branchitis-nodosa-type, the bacteria invaded the connective tissue of the gill filaments and caused the production of the granulation tissue in the acute case. The granulomas were formed to wall off each druse in the granulation tissue in the chronic case. Nodules consisting of macrophages and granulomas were formed at the metastatic lesions in visceral organs. 19) Based on the histopathological findings, types 1 ,2 and 3 could be recognized as one kind of manifestation type ; general abscess-nodule type. According to the above recognition, it was determined that N. kampachi infection had two manifestation types as the general abscess-nodule-type and branchitis-nodosa-type. N. kampachi was determined as a bacterium causing granulomatous inflammation. Total discussion 20) The author comparatively discussed infectious diseases caused by 14 species of fish-pathogenic bacteria. Based on the histopathological findings of them, it was determined that i) A. hydrophila was characteristic in the production of toxic substance, ii) V. anguillicida, V. anguillarum, Vibrio sp., Vibrio sp. RT group, Aeromonas salmonicida, Pseudomonas anguilliseptica and Flexibacter columnaris were characterized by a strong invasivness into the tissue and causing necrosis, iii) E. tarda was a pyogenic bacterium and iv) Streptococcus sp., N. kampachi, Pasteurella piscicida, Corynebacterium sp. and Mycobacterium piscium were bacteria causing granulomatous inflammation. 21) The author discussed characteristics of inflammatory responses in fishes. As for inflammatory responses concerned with defensive reactions, phagocytosis of neutrophils and macrophages and demarcation with the granulation tissue were not efficacious against E. tarda, P. piscicida, Streptococcus sp., Corynebacterium sp. and N. kampachi, because these bacteria were proven to be able to multiply in phagocytes and invade the granulation tissue. On the other hand, the granuloma formation was the most efficacious, defensive response against these bacteria, because i) granulomatous inflammation was manifested in the terminal stage after the defensive acts of neutrophils, macrophages and the granulation tissue were defeated, and ii) those bacteria markedly decreased in numbers after they were walled off by the layer of epithelioid cells.
三重大学水産学部の論文

著者

宮崎照雄
三重大学生物資源学部

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