牛のデルマトフィルス症に関する研究 Studies on the dermatophilosis in cows

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著者

    • 金城, 英企 キンジョウ, エイキ

書誌事項

タイトル

牛のデルマトフィルス症に関する研究

タイトル別名

Studies on the dermatophilosis in cows

著者名

金城, 英企

著者別名

キンジョウ, エイキ

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268号

学位授与年月日

1988-03-09

注記・抄録

博士論文

デルマトフィルス症は放線菌目のDermatophilus congolensisによって起こる牛・馬・めん羊・山羊・ 人などの表在性皮膚感染症であって,本菌は現在のところ偏性寄生(病原)体の一種に数えられている。 本症の自然感染発生例は世界的に確認されており,特に重症例はアフリカ大陸を中心に熱帯地域での発生が多数報告され,経済的な被害が深刻な状況にあると言われている。日本での確実な本症の発生は全くなかった。1978年,沖縄県先島諸島の放牧牛に急性または漫性経過をとる治療困難な悪性の表在性皮膚炎の集団的または散発的発生が認められたので,この代表的症例について微生物学的検索を実施した結果,Dermatophilus congolensisを分離・証明した。この症例は日本における牛デルマトフィルス症の初発例であり,本症は熱帯獣医学上重要な牛の皮膚疾患であり,わが国での蔓延も十分に考えられたことから,この野外自然発生例を中心にして,本症に関する研究に着手した。 本論文では,この本邦初発の牛デルマトフィルス症に関する臨床獣医学的所見と病理組織学的所見及び分離菌の微生物学的性状について報告するとともに,分離菌の抗原分析の結果,野外応用が可能となったDermatophilus congolensis診断抗原を用い,沖縄県下の飼育黒毛和種を対象にして抗体保有率を検索したので,その概要について報告する。1. 臨床獣医学的所見 1978年4月から1980年2月にかけて沖縄県先島諸島の4離島の放牧牛25頭に悪性皮膚炎の発生が認められたが,原因・治療法が不明であった。 罹患牛25頭の月齢は生後6か月以下のものが20頭(80%)で,この中には15日齢,20日齢の幼牛が含まれていた。さらに,本皮膚炎に敗血症を併発し,死亡する牛も見られた。本症の発生は5~8月が44%(11/25)とやや多かったが,本症の発生は3月以外常に見られた。発症牛は被毛不潔・栄養不良で発育遅延個体に多く,皮膚症状の特徴は滲出性の膿庖及び膿痂疹の形成であって,被毛の刷毛様・樹皮様所見と岩状痂皮の形成であったが,痒覚症状は示さない。感受性試験の結果から治療試験を実施したところ,Penicillin 8,000IU-Streptomycin 10mg(/Kg)の合剤4日連続筋肉内注射後,1,2,3,5,7,10日間隔で合計10回投与,Erythromycin 6mg/Kg 2日連続筋肉内注射は効果的であった。2. 病理学的所見 初期の皮膚病変は膿庖及び丘疹の形成で,局所は持続的な漿液の滲出とその凝固にともなう痂皮の形成が顕著であった。隆起した痂皮は厚さ1.5~2.0cmに達して容易に剥離し,その脱落創面は真皮で,表面は充血して赤くなり,その周辺からは漿液の滲出が見られ,イチゴ様であった。組織学的には滲出性表在性皮膚炎もしくは痂皮形成炎で,痂皮層・表皮層及び毛嚢の病変部には特徴的な菌糸様のGram陽性菌が多数見られた。表皮表面には不全角化細胞,若干の変性好中球,細胞核残屑及び漿液性滲出物からなる痂皮が表皮層に連続的にもしくは遊離して存在する。この痂皮及び表皮細胞間には多数の菌糸様菌体が認められた。毛嚢の外根鞘・外根鞘に著しい菌の増殖が認められる。3. 分離菌の微生物学的所見 分離菌は5%牛血液(馬血液)加 Brain Heart Infusion寒天培地での37℃・48時間・好気性培養でよく増殖し,微好気性培養で気中菌糸を形成する。好気性培養の集落は表面が不規則な隆起を示す直径0.5~1㎜の黄橙色で培地に固着し,白金耳での釣菌-移植には1集落全体を摘出した。Gram染色での形態は伸長菌糸はよく分岐し,横断隔壁を形成し,やがて縦断裂を生じて運動性のある球菌状集塊として確認された。 透過型電子顕微鏡での観察で叢毛性鞭毛と細胞表面にslime様構造物の存在が確認された。 分離菌の生化学的性状としてはカタラーゼ・ウレアーゼ陽性で溶血性があり,ゼラチン・カゼイン・デンプン分解性であったが,チロシン・キサンチンは分解されなかった。糖類の分解性ではグルコース・フラクトース・ガラクトース及びマルトースが陽性であった。これらの性状から分離菌はDermatophilus congolensisと同定された。 新分離菌のDermatophilus congolensis No.1株を用いて,熱抽出抗原・超音波処理抗原・TCA抽出抗原及び濃縮培養上清抗原を調製した。この4種抗原の蛋白量は,培養上清抗原:1.33mg/ml,TCA抽出抗原:0.28mg/ml,熱抽出抗原:0.21mg/ml及び超音波処理抗原:0.21mg/mlであった。寒天ゲル内拡散沈降反応による抗原力価は超音波処理抗原で64倍,培養上清濃縮抗原で16倍及び熱処理抗原・TCA抽出抗原で4倍の力価を示した。 沖縄県下の放牧牛で問題となるピロプラズマ病(Babesia bigemina,Babesia bovis,Babesia ovata,Theileria sergenti),アナプラズマ病(Anaplasma marginale,Anaplasma centrale),Papilloma(免疫血清),Dermatophilus感染牛及び非感染正常対照牛血清について,超音波処理抗原を用いた沈降反応によって使用抗原の特異性を検討した結果,本抗原はDermatophilus感染牛血清にのみ反応し,他の病原体などの感染牛血清には反応しなかった。 分離菌のウサギに対する感染試験では,肉眼的に確認できる痂皮様物が形成され,この痂皮のGiemsa染色で菌糸様菌体が鏡検され,5%馬血液加 Brain Heart Infusions寒天培地の37℃,48時間CO_2培養及び好気性培養によってDermatophilus congolensisが分離された。4. デルマトフィルス症の血清学的診断 超音波処理抗原(4単位)を診断用抗原として用い,本症罹患牛の抗体価を測定し,皮膚病変スコアと沈降素価・間接赤血球凝集素価及び酵素結合抗体免疫吸着アッセイとの関連性を検討した。 沈降反応の陽性限界を抗体価1:1以上とした場合,臨床的に皮膚病変が全く認められなかった85例中3例(3.5%)が陽性と判定された。一方,皮膚病変スコアI水準の個体21例中14例(66.7%)は沈降抗体陰性であったが,病変スコアII及びIII水準の症例ではいずれも抗体価1:2以上であり,病変スコアIII水準の8例中6例(75.0%)は抗体価1:32以上であった。 間接赤血球凝集反応の陽性限界を抗体価1:10とした場合,皮膚病変の全く認められなかった85例中40例(47.1%)が陽性と判定された。逆に皮膚病変スコアI水準の個体21例中3例(14.3%)は凝集抗体価1:10以下であった。一方,皮膚病変スコアII及びIII水準の症例ではいずれも抗体価1:10レベル以上であった。 酵素結合抗体免疫吸着アッセイ法の陽性限界を0D:0.500レベルとした場合,皮膚病変が全く認められなかった29例中2例(6.9%)(1,000倍希釈系)が陽性と判定された。一方,皮膚病変スコアI水準の症例19例中15例(78.9%)が陰性であった。病変スコアII及びIII水準の症例では全て陽性であった。 沈降素価と凝集素価との関係を解明する目的で,皮膚病変スコアとともに同一個体血清で両抗原抗体反応を実施した125例のデータに,病変スコア不明牛で両抗原抗体反応を実施した牛47例のデータを加えた合計172例の抗体価について分析した結果,両抗体価間の相関係数はr=0.898で,両反応成績はよく符合していた(P<0.01)。 沈降素価とELISA抗体価との関係をみるために皮膚病変スコアとともに同一個体血清で両抗原抗体反応を実施した64例のデータに,病変スコア不明牛血清で両抗原抗体反応を実施した11例を加え,合計75例の抗体価につき分析した結果,両者間の相関係数は被検血清の1,000倍希釈系でr=0.896で両反応はよく符合していた(P<0.01)。 沖縄県下の飼育牛についてDermatophilus congolensis抗体の保有状況を超音波処理抗原(4単位)を用いた寒天ゲル内拡散沈降反応によって検索した。全供試牛980頭中84頭(8.6%)が抗体陽性であった。島別の抗体保有率は沖縄諸島での成績は4.0%(16/405)で,その内訳は伊是名島0%(0/20),伊平屋島14.8%(9/61),沖縄本島2.2%(7/314),久米島0%(0/10)であった。大東諸島での陽性例は検出されなかった(0/26)。宮古列島での陽性率は16.2%(16/99)で,その内訳は来間島33.3%(1/3),多良間島15.6%(15/96)であった。八重山列島での陽性率は11.6%(52/450)で,その内訳は石垣島9.5%(22/231),竹富島0%(0/19),黒島23.7%(27/114),西表島0%(0/47)及び与名国島7.7%(3/39)であった。 以上の結果からDermatophilus congolensisの野外の抗体保有状況・感染牛の診断及び摘発などに,寒天ゲル内沈降反応・間接赤血球凝集反応及び酵素抗体法の三者は十分応用し得る成績を得たが,抗原精製・手技の簡易性,特に反応の特異性の点でゲル内沈降反応の応用が適当と結論された。

Dermatophilosis is a superficial dermatitis caused by Dermatophilus congolensis which belongs to Actinomycetales Buchanam and occurs in bovine, horse, sheep, goat or man. The Dermatophilus congolensis classified under the obligate parasites in the present time. The natural occurence of Bovine dermatophilosis is reported worldwide. Serious dermatophilosis is reported in the Tropical zone, especially in the Africa Continent. It inflicts great economical damage to stockowners. There have been no reported of such occurence in Japan. Acute, chronic or malingnant cutaneous symptoms characterized by incrustation and alopecia were notice in calves of the indigenous Japanese Black breed grazing on island of Sakishima ( which are located south-west ) of Okinawa Island. Organisms were isolated from lesions in these calves and were identified bacteriologically as Dermatophilus congolensis. This was the first case of dermatophilosis in Japan. This skin disease found in bovine is important in tropical veterinarism. As an epidemic in JAPAN is possible, I started reseach on this disease, focusing especially on this natural occurence. This paper deals with clinical, pathological and bacteriological examination on dermatophilosis. Diagnosis antigen with dermatophilosis was prepared throuth the serological study of Dermatophilus congolensis antigens. Using the Dermatophilus congolensis antigen, it made the selorogical diagnosis of dermatophilosis in naturally infected cattle possible. This paper deals with investigation of the present of Dermatophilus congolensis antibodies in cattle of the indigenous Blacks breed, grazing on Okinawa islands.1. Clinical veterinary findings Such cutaneous symptoms characterized by incrustation and alopecia were noticed in calves or the indigenous Japanese Black, grazing on four of the Sakishima Islands of Okinawa. It was a skin disease whose cause and cure were unknown. At the time of examination, 80% (20/25) of the cattle examined was less than 6 months of age. They included two calves, 15 and 20 days old. Moreover, some of the calves developed septicemia and this skin disease at the same time and died. 44%(11/22) of the disease occured during May and August. This occurance rate is slightly higher than in the average month but, the disease occurs throughout the year, except March. This disease was more likely in cattle with, unclear hair and-or under-developmen There was a marked defference in growth between the infected calves and the healthy nomal onens of the same age. The cutaneaua symptom were characterized by the formation of the diapedetic pustule and crust. Paint-brush, bark and rock form were found on the skin of infected cows as various macroscopical forms of crust. No infected Animals were affected with pruritus. Medical treatment examinations were carried out based the results of sensitivity tests of naturally infected cattle. Satisfactory therapeutic effect was attained by intramuscular injections, of a combination of 10mg/kg streptomycin and 80000U/kg of benzyl penicilin, for 4 consecutive days, followed by 1, 2, 3, 5, 7, and 10 days intervals Also 4mg/kg of erythromycin for 2 consecutive days was effective.2. Histopathological findings Initial changes were, formation of vesicles; papillary projections; and pustule exudation from these structures, and incrustation. The crust were scattered and formed blocks 1.5-2 cm thick. The stale crust were readily detached from the skin by the finger. The detached part revealed the corium, and hyperemic with diapedeti blood on its border. It had a strawbery-like apperance, and lacked hair. Histopathological examination revealed exudative superficial or inflammation with incrustation. Many characteristic Mycelia of Dermatophilus congolensis were present in the crust, the epidermis layer and the hair follicle. The crust composed of insufficiently conified cell, some degenerative neutrophilu neuclear debris and serous exudate was present in the epidermis layer, and It was clustered together or scattered about. Many branching mycelia were noticed among the crust and epidermal cells. A remarkable bacterial multiplication was seen in the outer and inner roof sheath of the hair follicle.3. Microbiological examination of isolated organisms The isolated organisms grew in 48 hours, under aerobic conditions in a brain heart infusion agar to which 5% bovine blood or 5% equine blood had been added. The organism formed aerial hypha in a box containing 10% CO2. Colonies yellowhish in color were formed. They were 0.5-10 mm in diameter and had irregular projections on the surface. They were so hard that only the whole colonies could be picked up a loop. Gram-stain form with Stereomicroscopy of the colonial border revealed the development of hyphae only in the anaerobic culture. The thick filament made good hyphal development and contained coccoids arranged transversely. The thick filament was divided into thin elements, which developed to crossed logitudinally and latitudinally with one another. Organism in 48 hours culture on agar, or in liquid medium were all motile. The presence of flagella and slime like stracture confirmed on cell walls by trans mission electron microscopy.4. Biochemical reactions The isolated organisms was catalase-positive as determined by a tube test. The urease was regulary positive. They hydrolyzed stach, and casein but not tyrosin and xanthin. Gelatin was liquefied by almost all strains. They decomposed glucose fructose, galactose and maltose. The isolated organisms were identified as Dermatophillus congolensis by these biochemical reactions.5. Preparation of antigen Dermatophilus congolensis antigens were prepared by the No-1 strain of isolated Dermatophilus congolensis. There were four antigen, namely, heat treated antigen, sonicated antigen, trichloro-acetic acid extract antigen and condenced supernatant fluid antigen. The protein quantity of antigen were 1.11mg/ml in condenced antigen, 0.28mg/ml in TCA extracted antigen, 0.21mg/ml in heat treated antigen and 0.21mg/ml in sonicated antigen. Agar gel diffusion of the antigen titer induced 64 unit in the sonicated antigen, 16 unit in the condenced antigen and 4 unit in heat treated and TCA extracted antigen. As piroplasmosis and anaplasmosis become a subject discussion in grazing cows on Okinawa islands, Antibodies were collected from cows infected with Babesia bigemina, Babesia vobis, Babesia ovata, Theileria sergenti occuring in Piroplasmosis and Anaplasma marginale, Anaplasma centrale occuring in Anaplasmosis, and Papilloma immune serum. Naturally infected Dermatophilus congolensis serum was supplied as the positive serum, and the healthy cow serum was used as the negative serum. The Dermatophilus congolensis antigen responded only to the naturally infected Dermatophilus congolensis serum and did not react to the other diseased cow serum.6. Experimental infection In the infectious examination crust like structures visible to the naked eye were formed on the rabbit. Hyphae was found in the crust on the skin of the infected rabbit by Giemsa stain, throuth microscope. The isolation medium used was brain heart infusion agar to which 5% equine blood had been added. The plates were incubated under aerobic conditions and in box contaning 10% CO2. The Dermatophilus congolensis was isolated from the crust on the skin of the infected rabbit.7. Serological diagnosis on Dermatophilosis 4 unit of the sonicated antigen was employed as the diagnostic antigen. It was used to detect the antibody titer of the infected cows with dermatophilosis, to see whether the score of cows infected with dermatophilosis had correlation with the precipitation titer, the indirect haemagglutination titer and the enzyme-linked immunosorbent assay. Although the skin disease could not diagnosed on 85cows clinically, there were 3/85 cows (3.5%) that more positive when the positive limit was decided at a minimum antibody precipitation titer of 1:1. On the other hand, 14 of 21 cases in the score 1 categorie was antibody precipitation titer negative but, the precipitin titer was over 1:2 unit in the score 2 and 3 case, and was over 1:32 unit in 6/8 cows in the score 3 case. Although the skin disease could not be diagnosed on 85 cows clinically, there were 40/85 (47.1%) cows that were positive when the positive limit was decided at a minimum of 1:10 unit in the indirect haemagglutination. Convensely, the indirect haemagglutination titer was below 1:10 unit in 3/21(14.3%) cows in the score 1 case. As compared to 1:10 unit in the score 2 and 3 case. Although the skin disease could not be diagnosed on skin of cows clinically. there were 2/29(6.9%) cows that were positive with 1000 dilution of serum when the positive limit was decided on optical density of (260nm) 0.500 in the enzyme-linked immunosorbent assay. There were negative 15/19(78.9%) in the score 1 case. All were positive without exception in the score 2 and 3 case. As a result of analysing the antibody titer of 172 case - the data of 125 were cases where both the precipitation and indirect haemagglutination tests were performed with the serum of known score; and the data of 47 were cases where the precipitation and haemagglutination tests were performed with serum of unknown scores - in order to clarify the relationship between the precipitin titer and passive haemagglutinin titer, the correlation between the two antibodies was γ=0.898, thus the two reaction results matched well (P<0.01). As a result of analysing the antibody titer of 75 cases - the data of 64 were cases where both the precipitation test and enzyme-linked immunosorbent assay were performed with the serum of known score; the data of 11 were cases where both the precipitation test and enzyme-linked immunosorbent assay were performed with serum of unknown scores - in order to clarify the relationship between the precipitin titer and enzyme-linked immunosorbent assay titer, the correlation cofficient between the two antibodies was γ=0.896. Thus both the precipitation test and enzyme-linked immunosorbent assay reaction results matched well in 1000 dilution (P<0.01).8. Survey for the presence of precipitins to Dermatophilus congolensis By the precipitation test, Dermatophilus congolensis antibodies were found in sera from the indigenous Japanese Blacks grazing in Okinawa Islands using 4 units of sonicated antigen. Antibody was found in 8/6% of the sera from 980 cows collected at random. The percentage of antibody holders were 0% of 20 in Izena, 14.8% of 61 in Iheya, 2.2% of 314 in mainland of Okinawa, 0% of 10 in Kume, 0% of 13 in Minamidaito, 0% of 13 in Kitadato, 33.3% of 3 in Kurima, 15.6% of 96 in Tarama, 9.5% of 231 in Ishigaki, 0% of 19% in Taketomi, 23.7% of 114 in Kuro, 0% of 47 in Iriomote, 7.7% of 39 in Yonaguni, and 4.0% 0f 405 in Okinawa Islands, 0% of 26 in Daito Islands, 16.2% of in Miyako Islands and 11.6% in Yaeyama Islands. From the previous results, the precipitation, indirect haemagglutination and enzyme-linked immunosorbent assay tests were shown to be useful for diagnosing and surveying cattle for Dermatophilosis. Althogh the precipitation test would be useful in that precipittinogen can be prepared more easily than enzyme-linked immunosorbent assay and indirect haemagglutination antigens, the precipitation test technique is easier than the enzyme -linked immunosorbent assay or indirect haemagglutination, and is more specific..

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