内毒素による壊疽性乳房炎に関する研究 Endotoxin-induced gangrenous mastitis in cows

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著者

    • 凾城, 悦司 ハコギ, エツジ

書誌事項

タイトル

内毒素による壊疽性乳房炎に関する研究

タイトル別名

Endotoxin-induced gangrenous mastitis in cows

著者名

凾城, 悦司

著者別名

ハコギ, エツジ

学位授与大学

麻布大学

取得学位

獣医学博士

学位授与番号

乙第272号

学位授与年月日

1988-06-01

注記・抄録

博士論文

乳牛の壊疽性乳房炎は,近年飼養規模の拡大に伴い,飼養管理の省力化ならびに抗生物質の頻用による菌交代現象によって多発の傾向にある。とくに,本性は甚急性の経過をとり,急激な全身症状と乳房の局所症状を伴い,そのほとんどのものが治療効果がなく,死亡および廃用の転帰をとるため,家畜衛生上重要な問題を提起している。 しかし,本症の実態および発生機序については今なお不明な点が多く,原因菌の検出状況,血液生化学的所見についても系統的な研究が少なく,未だに議論が多い。 これらのいわゆる壊疽性乳房炎の発生機序については,臨床症状,血液学的および病理組織学的所見などから,グラム陰性桿菌による内毒素中毒が疑われている。 このようなことから,筆者は家畜における内毒素の基礎的研究を行うとともに野外で発生した壊疽性乳房炎について内毒素の検出を試みたところ,本症の多くはグラム陰性桿菌による内毒素中毒であることを明らかにした。 さらに,本症の発生機序を探るために臨床的,細菌学的,病理学的,血液生化学的検査を実施し,発生機序についての検討を行った。 本研究の概要は次のとおりである。1.発生状況 1974年から1984年までの11年間における兵庫県淡路地方について,壊疽性乳房炎の発生状況を調査した。発生総数は794頭で総死廃頭数16,142頭の4.9%,乳房炎死廃頭数2,069頭,38.4%を占めており,年々増加する傾向がみられた。 発症時期は分娩と密接な関係があり,分娩後7日以内に81.3%(204/251頭)のものが発病し,経過が急激であるため発症後3日以内に78.5%(197/251頭)のものが死亡あるいは廃用の転帰をとっていた。 主要な症状として,元気消失,食欲廃絶,心悸亢進,起立不能,悪臭のある下痢などの全身症状と乳房の紫斑,冷感,腫大,乳汁の変化が見られた。2.細菌学的検索 1974年から1984年までの11年間に臨床的に壊疽性乳房炎と診断したホルスタイン種乳牛65頭,急性乳房炎牛12頭および異常の認められない健康対照牛26頭について,細菌,病理,血液ならびに内毒素の検索を行った。 壊疽性乳房炎発症牛の乳汁の細菌検査では,Escherichia coli 50.8%(33/65),Pseudomonas aeruginosa 16.9%(11/65)とこの両者の菌種が主に分離され,次いで Klebsiella pneumoniae 4.6%(3/65),Enterobacter aerogenes 4.6%(3/65),Alcaligenes faecalis 1.5%(1/65),Acinetobacter anitratus 1.5%(1/65)などグラム陰性桿菌が全体の80.0%を占めていた。これに対しグラム陽性菌は全体の20.0%(13/65)であり,Bacillus cereus,Clostridium perfringens,Streptococcus sp.,Actinomyces pyogenes,Staphylococcus aureus などが分離された。 主要臓器の細胞培養では,罹患分房からはほぼ純粋に起因菌を分離したが,その他の臓器のうち肝,脾,腎などからはほとんど分離出来なかったことから本症の発生機序は起因菌の菌体毒素の作用によることを確認した。 また分離菌の血清型別では,E.coli24株について0群別を行ったところ02が5株,08,075,088がそれぞれ2株ずつ,09,064,076,089が1株ずつ,型別不能9株であった。P.aeruginosa 22株の血清型別はGが6株,E,F,Iがそれぞれ3株ずつ,Aが2株,B,Kが1株ずつであり,両菌種ともにほぼ限られた血清型のパターンがみられた。このことから糞便あるいは腸管由来のこれらの細菌が,内因的もしくは外因的な感作を受けて感染し,さらに発病に至ったものと考えられる。3.病理組織学的検索 病理解剖所見では,罹患分房を剥皮すると皮下織は顕著な浮腫,点状出血が認められた。割面は暗赤色もしくは淡褐色を呈し,強い出血と間質の浮腫が広い範囲でみられ,健康部と病変部との境界は明瞭であった。主要臓器では,肝はやや腫大し,淡黄色脂肪化ならびにうっ血が認められた。 腎には充血と一部点状出血がみられ,脾は腫大し脾柱不明瞭で,肺および心において暗赤色斑が認められた。 病理組織所見では,乳房腺胞内の出血,腺胞上皮細胞の変性壊死,水腫,血管の線維素性壊死が認められた。また,血栓形成は罹患分房に最も多くみられ,次いで肝,腎,肺の順に認められたことは,ヒトの内毒素血症にみられる血管内凝固症候群ときわめて類似する所見であった。主要臓器の病変では,肝の脂肪変性,出血,壊死,血栓形成,腎の出血,尿細管上皮細胞の変性壊死,肺のうっ血および出血,心の小壊死巣がみられた。このことから,細菌の毒素により直接的に組織障害を引き起こし,重篤な症状を呈するものと考えられた。4.血液生化学的検索 壊疽性乳房炎牛では,赤血球数,ヘマトクリット値の増加,白血球数の減少,好酸球,分葉核好中球および単球の減少と桿状核好中球の増加が認められた。とくに,分葉核好中球の減少は罹患分房内への大量の移動と内毒素の刺激による好中球の辺縁趨向によるものと考えられる。 また,血清総蛋白量,アルブミン,グロブリンおよび血清カルシウムの減少と血清尿素窒素,血清無機リンの増加が認められた。血清酵素量では,GOT,GPT,LDHは有意に高く,ALP,γ-GTPも全般的に高値であった。これは罹患した乳腺組織および主要臓器中でのこれらの酵素活性値が著しく低下しており,内毒素による臓器障害,とくに乳腺組織の破壊によりこれらの酵素が血液中に逸脱したためと考えられる。 血液凝固系の検査では,血小板数の減少,プロトロンビン時間,活性部分トロンボプラスチン時間の延長,血漿フィブリノーゲンの著減が認められたことは,ヒトの内毒素血症にみられる血管内凝固症候群ときわめて類似する所見であった。5.内毒素の検索 本症牛の発生機序の解明ならびに予後診断の目的で内毒素の基礎的検討を行った。健康牛の血漿および乳汁中には合成基質を直接水解する非特異的アミダーゼ活性が存在していることが判明したため,PCA前処理を行いリムルス反応影響因子を除去し,合成発色基質法により,血漿および乳汁中の内毒素を高精度に再現性よく定量する方法を確立した。 そこで,健康牛(n=36)の血漿および乳汁中の内毒素量を測定したところ,すべて10pg/ml^-1以下であった。また,急性乳房炎牛でグラム陰性桿菌が分離された症例(n=12)は,血漿中の内毒素量はすべて10pg/ml^-1以下であったが,乳汁中では1,147.0±766.8pg/ml^-1と高い値を示した。しかし,グラム陽性菌が分離された症例(n=6)は,血漿および乳汁中の内毒素量はすべて10pg/ml^-1以下であった。 壊疽性乳房炎牛のうちグラム陰性菌感染群(n=30)の血漿中の内毒素量は,89.6±68.2pg/ml^-1と高値を示した。乳汁中の内毒素量は血漿中のそれよりも著しく高く9.1±5.2×10^6pg/ml^-1であったことから,グラム陰性菌群(感染)による本症は内毒素血症によるものであることが確認された。一方,グラム陽性菌感染群(n=3)は,血漿および乳汁中の内毒素量はすべて10pg/ml^-1以下であった。 また,本症発症牛の内毒素陽性牛は陰性牛に比べて,重度な臨床症状を示すものが多く,廃用までの日数が短い傾向がみられた。また,内毒素によってのみ活性化される因子に合成発色基質を加えたエンドスペシーテストで検討したところ,きわめて高い特異性で内毒素が検出されたことから,本症牛のうちグラム陰性菌感染群の発生要因は,内毒素によることが再確認された。 このように合成発色基質法を用いたリムルステストにより本症牛のうち,グラム陰性菌感染群の血漿および乳汁中から高濃度に内毒素が検出されたことから,グラム陰性桿菌による急性および壊疽性乳房炎の早期診断となり,さらに効果的な治療法と予後判定に有用な指標となると考えられる。 以上に述べた成績から,グラム陰性菌群による壊疽性乳房炎の発生機序は,以下のとおり結論づけられた。 本症は,分娩直後に多発し,病性が急激なため治療を施す余裕もなく死亡および廃用の転帰をとるものが多い。感染経路として,主に乳頭口から侵入したグラム陰性桿菌が,乳腺乳槽および乳管内で増殖し,その結果,大量の内毒素を産生する。これら産生された内毒素が乳腺上皮から循環血液中に流入して血液凝固線溶系や補体系へ作用し,血管内凝固症候群を引き起こす。また,内毒素の直接的な作用により乳房および主要臓器の出血,壊死,血栓形成などの組織障害を起こすとともに,全身性シュワルツマン反応が加わって,死亡するものと考えられる。 さらに,本症の発生誘因として,分娩によるストレス,飼養環境の不良,高栄養の飼料給与による脂肪肝,代謝障害,潜在性乳房炎など母体に病原菌の侵入を容易にするような条件が加わったことも十分考えられる。

The scaling up of daily cow husbandry in recent years has naturally demanded a labor saving in its management and abuses of antibiotics have led to the increased incidence of microbial flora substitution in animal. Under these circumstances, gangrenous mastitis in dairy cows has been found to occur more frequently in many areas. This desease is characterized by a sub-acute course with both systemic symptoms and local symptoms of the udder resulting in the disuse or death of affected animals.Thus, this disease is now considered to be a serious problem for livestock hygiene. At present, however, many aspects concerning the actual conditions and the developmental mechanisms of this disease are obscure, and few systematic studies have reported on the detection of the bacteria or the hemato-biochemical findings. Accordingly, there is still considerable argument of this subject. On the basis of only the clinical symptoms and the hematological or histopathological findings, it is assumed that gangrenous mastitis may be related to intoxication induced by endotoxin of Gram-negative bacilli. The author preformed a fundamental study of endotoxin in livestock, while attempting to detect endotoxin in gangrenous mastitis cases occurring in the field. It was found that, in most cases, the disease was induced by intoxication associated with endotoxin of Gram-negative bacilli. In addition, clinical, bacteriological, pathological and hemato-biochemical examinations were carried out for the purpose of investigating the developmental mechanisms of the disease. The results are summarized below.1. Incidence The incidence of gangrenous mastitis was investigated for 11 years between 1974 and 1984 in Awaji, Hyogo prefecture. The total incidence was 794, which corresponded to 4.9% of 16, 142 deaths in total and 38.4% of 2,059 deaths due to mastitis. There was a yearly increase in numbers. The time of incidence was closely related to calving. Of 251 cases, 204(81.3%) showed the symptoms within 7 days after calving and 197 out of 251 cases (78.5%) died or were put out of use within 3 days after manifestation of the symptoms due to the rapid course of the disease. The main symptoms included systemic symptoms such as lethargy, anorexia, cardiopalmus, astasia, and diarrhea with malodor, and local symptoms of the udder such as purpura, cold sensations, swelling of the udder and changes in milk quality. 2. Bacteriological Investigation Bacteria, pathology, blood and endotoxin were investigated for 11 years between 1974 and 1984 in 65 Holstein cows clinically diagnosed with gangrenous mastitis, 12 cows with acute mastitis, and 26 healthy cows as controls. Bacteriological examinations of milk of the cows with gangrenous mastitis revealed the following Gram-negative bacilli : Escherichia coli in 33 out of 65 cases (50.8%) ; Pseudomonas aeruginosa in 11 out of 65 (16.9%) ; Klebsiella pneumoniae in 3 out of 65 (4.6%) ; Enterobacter aerogenes in 3 out of 65 (4.6%) ; Alcaligenes faecalis in 1 out of 65 (1.5%) ; and Acinetobacter anitratus in 1 out of 65 (1.5%). This indicates that Gram-negative bacilli accounted for 80.0% of all cases. The following Gram-positive bacteria accounted for 20.O% (13/65) : Bacillus cereus ; Clostridium perfringens ; Streptococcus sp. ; Actinomyces poygenes ; and Staphylococcus aureus. In cultures of bacteria obtained from the main organs, the etiological bacteria were isolated in an almost pure state from the affected udder, but could be hardly isolated from the liver, the spleen or the kidney. From the results, it was concluded that the disease is caused by the action of endotoxins of the etiotogical bacteria. According to the 0 serotype classification, 24 strains of isolated E.coli were categorized as follows : 5 strains in 02 ; 2 each in 08, 078 and 088 ; 1 each in 09, 064, 076 and 089 ; and 9 strains could not be evaluated. On the other hand, the serotype of 22 strains of P.aeruginosa was classified as follows : 6 strains in G ; 3 each in E, F and I ; 2 in A ; and 1 each in B and K. Since both species showed rather limited serotypes in this manner, it was considered that these bacteria, derived from feces or the intestinal tract, are activated by intrinsic or extrinsic sensitization to induce the disease. 3. Histopathological Investigation Pathologic autopsy revealed marked edema and petechial hemorrhage in the subcutaneous tissue by decorticating the affected udder. The incised surface had a dark red or light-brown color with a severe hemorrhage and edema of the interstitial tissues over an extended area, and the lesion was clearly separate from healthy areas. The findings of the main organs were as follows : the liver was slightly swollen and showed pimelosis of a light yellow color and congestion ; hyperemia and partial petechia were observed in the kidney ; the spleen was swollen showing an unclear substrate ; and dark red spots were recognized in the lung and the heart. Histopathological findings revealed hemorrhages inside the glandular alveoli of the udder, degenerative necrosis of epithelial cells of the alveoli, and edema and fibrinous necrosis of the blood vessel. Thrombus formation was observed in the affected udder, the liver, the kidney and the lung, in this order of frequency, which is a very similar finding to that of intravascular coagulation syndrome of endotoxemia in man. Lesions of the main organs were as follows : fatty degeneration hemorrhage, necrosis and thrombus formation in the liver ; degenerative necrosis of epithelial cells of the uriniferous tubule in the kidney ; congestion and hemorrhage of the lung ; and small necrotic lesions of the heart. Thus, it was considered that endotoxin directly causes tissue damage, leading to severe symptoms. 4. Hemato-biochemical Investigation The cows with gangrenous mastitis showed increases in red blood cells, hematocrit and stab leucocytes, and decreases in white blood cells, eosinophils, filamented neutrophils and monocytes. A decrease in filamented neutrophils, in particular, is concidered to be due to their transfer in a large amount into the affected udder and the margination of neutrophils stimulated by endotoxin. There were decreases in total serum protein, albumin, globulin and calcium, and increases in serum urea nitrogen and inorganic phosphorus. Concerning serum oxygen, values of GOT, GPT and LDH were significantly high, while those of ALP and γ-GTP were rather high in general. In contrast, the activity of these enzymes was considerably decreased in the affected mammary gland tissues and the main organs. These enzymes may have been released into the blood following endotoxin-induced damage of the organs, particularly destruction of the mammary gland tissues. In tests of the blood coagulation system, the following results were obtained : a decrease in platelet count ; prolongation of prothrombin time and partial active thromboplastin time ; and a marked decrease in plasma fibrinogen. These results are very similar to the findings of intravascular coagulation syndrome of endotoxemia in man. 5. Investigation of Endotoxin A fundamental study of endotoxin was carried out in order to clarify developmental mechanisms and prognosis of gangreous mastitis in cows. The finding that plasma and milk of healthy cows have nonspecific amidase activity, which directly hydrolyzes synthetic substrates, allowed the author to establish a new, highly precise and reproducible technique for measuring endotoxin in plasma and milk. This technique employs a synthetic coloring substrate method, following the removal of factors affecting limulus reaction by pretreatment with PCA. Using the new technique, the quantity of endotoxin was measured in healthy cows (n=36), all of which showed 10 pg ml^-1 or less. In the cows with acute mastitis from which Gram-negative bacilli were separated (n=12), endotoxin in plasma was always detected at 10 pg ml^-1 or less, but it was found in milk at high concentrations of 1,147.0 ± 766.8 pg ml^-1. However, in all cases from which Gram-positive bacteria were separated (n=6), endotoxin concentrations in both plasma and milk were 10 pg ml^-1 or less. Among the cows with gangrenous mastitis in the Gram-negative bacterial infection group (n=30), endotoxin, concentrations in plasma showed a high value of 89.6 ± 68.2 pg ml^-1, while those in milk reached a very high value of 9.1 ± 5.2 × 10^6 pg ml^-1. This stongly suggested that the disease induced by Gram-negative bacterial infection is associated with endotoxemia. In the Gram-positive bacterial infection group (n=3), endotoxin in plasma and milk was always detected at 10 pg ml^-1 or less. The endotoxin positive cases with gangrenous mastitis tended to show severe clinical symptoms and a short period before disuse, compared with the negative cases. In addition, endotoxin was detected with high specificity by the endospecie test, in which only those parts of the path activated by endotoxin were reconstructed and a synthetic coloring substrate was added. This finding thus confirmed that endotoxin was a developing factor of the disease in the Gram-negative bacterial infection group. Since endotoxin was detected at high concentrations in plasma and milk of the Gram-negative bacterial infection group by the limulus test using the synthetic coloring substrate method, endotoxin may be considered to be a useful marker for early diagnosis of acute and gangrenous mastitis, leading to more effective therapies and more promising prognoses. These results led to the following conclusions concerning the dovelopmental mechanisms of gangrenous mastitis caused by Ggam-negative bacilli. In many cases, gangrenous mastitis occurs immediately after calving resulting in disuse or death due to its rapid course without allowing the sufficient treatment time. First, Gram-negative bacilli invade from the papilla into the cisternae of the mammary gland and the milk duct, where they proliferate and produce a large amount of endotoxin. The endotoxin flows out of the epithelia of the mammary gland into the circulating blood and induces intravascular coagulation syndrome by affecting the fibrinolysis system of blood coagulation and the complement system. Furthermore, it is considered that a direct action of the endotoxin causes tissue damage such as hemorrhage, necrosis and thrombus formation in the udder and the main organs, leading to systemic Shwartzman phenomena and, finally, death. In addition, the following conditions for the easy invasion of bacilli into the mother's body may be properly cited as non-endotoxin developmental factors : stress caused by calving ; poor rearing environment ; fatty deposits in the liver associated with a high content of nutrients in the feed ; metabolic disturbances ; and latent mastitis.

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