@misc{oai:az.repo.nii.ac.jp:00003236,
 author = {馬渕, 貞三 and Mabuchi, Teizo},
 month = {2013-02-19, 2014-08-18},
 note = {Chalk(brood) disease in honeybees, otherwise called chalk-brood, is an infectious disease caused by Ascosphaera apis (Ascomycota, Plectomycetes, Ascosphaeraceae). It infects honeybee broods(larvae and pupae), and white chalk-like mummification of dead larvae is its characteristics. In beekeeping, the incidence of Apis meillifera larvae infections is the major issue.
 In 1913, Maassen of Germany first reported this white mummification disease of larvae as "Kalk Brut." Then, in 1916 the causative fungus was named Pericystis apis. Claussen conducted detailed study in lifecycle of fungus originated from fungus-infected beebroods. He elucidated that the multinucleated reproductive organ synascus is formed by antheridium developing fertilization tube stretched to ascogonium and fertilizes. Identifying this sexual reproduction, he supported the classification of this fungus to Pericvstis apis of Ascomycota. Later, detailed mycological observation of this fungus was conducted by Spltoir and Olive (in 1955), and they reclassified and named this fungus Ascosphaera apis(A. apis).
 Currently, sporadic incidences of chalk disease are reported world wide, and the various studies on the control measure are being conducted. Improvement on beekeeping technique, production of hybrid species for disease resistance, selection of queen, development of microbicidal drug, synthesis of fungicide, and preventing complication with other disease are investigated. However, no decisive method of prevention is yet to be found.
 In Japan, Udagawa et al. reported findings of A. apis in imported honey from Canada and honey produced in Akita prefecture. Another reports of isolating A. apis from honey, collected in Tottori and Akita prefectures, were made by Yamazaki when studying microorganism distribution in honey. Despite these findings, no incidence of chalk disease of honeybee was reported.
 Thus, the research in chalk disease of honeybee in actual state of incidence and observation on the course of disease in Japan was far from being sufficient. However, chalk disease is added to the list of "reported communicable diseases" by 1997 "Amendment on infectious disease prevention act of domesticated animals" in Japan. The investigation was needed on this disease as the market demand and import of honey increase, and quarantine operation was required.
 The author found the natural incidence of chalk disease through animal hygiene practice, and had opportunities to conduct basic research needed to establish the hygiene guidelines for the prevention of this disease.
 This thesis is composed of five sections: 1) Findings of naturally occurred chalk disease, 2) Distribution status of A. apis, 3) Pathogenicity of isolated A. apis, 4) In vitro fungicidal effect of microbicidal drug on A. apis, 5) Investigation and research on A apis field prevention. Their outlines are reported as following.

1. Naturally Occurred Incidence of Honeybee Chalk(brood) Disease.
 On March 1979, three colonies out of 100 bee colonies transported from Kyushu Island to Gifu prefecture was found to contain abnormal honeybee larvae, which was a case of natural chalk disease incidence. Larvae were colored white, black, and dark brown, and were mummified. The numbers of such larvae were found in brood cells in the combs and hive entrance. The hives strength was weakened, and only few worker bees were found around the queen.
 In histopathological observation of the disease, from the cuticular layer of mummified larvae, large number of fungus was identified by the red color using PAS staining. Rod-shaped and septate hyphae are sometimes branching in Y shape. Formations of ascomata(cleistothecia) are also observed.
 In pathogenic examination of chalk disease, no bacterium was isolated by either blood agar medium or egg yolk agar medium. However, on fungus media, white cotton-like fungal growth was identified from the third day of culture, and from its morphological observation of its hyphae and ascomata(cysts), it is presumed to be A. apis.
 The isolated fungus has high sugar demand, and after 1 week culture on M40Y agar medium, thin membranous ascomata(cleistothecial cyst), spherically shaped asci(spore ball), and oval shaped ascospore were formed. Thus it was finally classified morphologically as A. apis in Ascomycota.
 This is the first reported case of naturally occurred chalk(brood) disease. Around the year of 1979, summer time temperature was continuously above 30°C throughout Japan, and it is speculated that such weather may have contributed to the incidence of this mycosis.

2. Distribution Status of Ascosphaera apis.
 The distribution of this fungus was investigated in honey-bee larvae kept by beekeeper, honey, and pollen. As the result, 16(25.0%) out of 64 beekeepers had identifiable white mum-mified larvae. A. apis were isolated from 80 colonies(25.0%) out of 320. This fungus was isolated from seemingly healthy larvae in the honeybee colonies identified with mummified larvae.
 A. apis were isolated from honey produced by 16 colonies(5.0%) of 10 keepers out of 320 colonies of 64 keepers. From pollen, the isolated rate was 3 positive cases(4.7%) out of 64 keepers.

3. Pathogenicity of lsolated Ascosphaera apis.
 Pathogenicity of this fungus was investigated by spraying spore solution to healthy larvae and observed for the infection status. Symptoms' rate of appearance increased in positive correlation to the spore concentration. When the spore concentration(sprayed 50mL/a comb-side) was 10^3/mL, the incidence rate was 40.0%; 10^4/mL, 55.0%; and 10^9/mL, 93.0%.
 The larvae have high sensitivity to the disease up to 4～5-day old. However, the larvae infected before 3-day old were either liquefied when dead or removed by adult bee, so the incidence of chalk disease was not identified.
 The rate of collection of inoculated fungus was complete in artificial infection testing, and A. apis was collected from white mummified larvae as in the pure culture.

4. In Vitro Fungicidal Effect of Antimicrobial Drugs on Ascosphaera apis.
 In order to test for fungicidal effect on A. apis, each diluted preparation were brought into contact to A. apis spore for 2.5, 5, 10, and 15 minutes at 20°C, then cultured in M40Y medium. Observation was made for the growth of fungus, and evaluated with the presence and absence of growth. As the result, among the antiseptic, invert soap had fungicidal effect at 15 minutes contact of 1,600 x dilution, amphoteric soap at 10 minutes of 800 x dilution, and iodine preparation at 15 minutes of 3,200 x dilution.
 Among 14 fungicides, sodium propionic acid(PNa) had MIC of 0.04～1.25mg/mL, which was relatively low in concentration.
 Microbiocidal gas, formaldehyde(FA:formalin 40 mL/m^3, 22°C) and ethylene oxide gas(EO: 10%, 30°C) did not show sufficient microbicidal effect at 0.5 and 1 hour contact, but 100% fungicidal effect was observed in 24 hours contact.

5. Prevention of Chalk Disease
 Antiseptic and erradicative effects of drugs were tested on artificially infected combs in frame, which was prepared three days prior to test with A. apis spore solution(50 mL of 10^9 spores/mL solution per one side of comb). After 8 days from spraying invert soap(800 x dilution, 100 mL per one side of infected comb), occurrence of mummified larva was completely lost, and the procedure was recognized as effective. Also, PNa(0.5% solution, 100 mL per one side of infected comb) was found to be completely effective in prevention after 8 days of drug spraying. No side effect was observed for both drugs in adult bees, larvae, and eggs.
 In residual drug testing on honey collected from test honeybee group, residual invert soap(1.4 ppm) was detected up to 3 days from spraying, but fell below detection limit on 4th day, suggesting the possibility of application to field use. Residual amount of PNa was 5,250 ppm on first day, but fell below detection limit(25 ppm) on second day and after.
 Fungicidal effect of EO gas was investigated on A. apis artificially infected comb(3 ×10^8/mL spores, 10 mL per one side of comb, and 48 hours culture in contact at 25°C). As the result, 100% fungicidal effect was observed in contact temperature at 20°C and over 6 hours contact to EO gas concentration of over 2～3%. At 30°C, it was 6 hours contact to 1~3%, and 2 hours contact to 5%.
 Eradication test for naturally infected combs are conducted on 9×15 cm cutting of infected comb submerged in testing antiseptic solution for 24 hours, and culture in M40Y agar medium to test for the survival of fungi. As the result, 100% eradicative effect was observed in 100, 200, 400, and 800 x dilutions, but 1,600 x dilution was not effective. Iodine preparation was effective at 1,600 x dilution, but the residual odor was too strong and determined unsuitable for actual use. The effect of microbiocidal gas was 88.9%(8/9) for FA gas(previously mentioned) for 24 hours exposure at 24°C, and 100%(9/9) for EO gas for 24 hours exposure at 30°C.

 On March 1979, three colonies out of 100 honeybee colonies transported from Kyushu Island to Gifu prefecture was found to contain mummified larvae of white or black color. In pathogenicity test, white cotton-like isolate of thread shaped fungus was obtained on fungal medium. And after two weeks of culture, formations of ascoma(cleistothecium), ascus(spore ball) and ascospore were identified, and it was the first identified chalk disease in Japan.
 Using the isolated fungus, the artificial infection of healthy honeybee colony was successfully conducted. As the result of investigating for the control measure, invert soap and amphoteric soap were found to be effective, and among the fungicides, PNa was effective. Also as the preventive measure for empty comb, disinfecting by FA gas and EO gas were effective., 蜜蜂のチョーク病chalk (brood) diseaseはチョークブルードchalk-broodとも呼ばれ、蜜蜂の蜂児(幼虫・蛹)がAscosphaera apis (Ascomycota子嚢菌門、不整子嚢菌類Plectomycetes、Ascosphaeraceae科の菌糸型真菌)によっておこす感染症であって、死亡幼虫の白墨状ミイラ化が特徴とされている。養蜂上では特にApis melliferaセイヨウミツバチの幼虫感染が最も重要である。
　この蜂児の白色ミイラ化する疾病については、1913年にドイツのMaassenにより"Kalk Brut〔白墨病〕"として最初に報告され、1916年に本症の原因真菌はPericystis apisと命名された。Claussenはカビ着生蜂巣に由来する真菌の生活史について詳細に検討し、多核性の生殖器官は造精器が受精管を伸長し造卵器に受精して受精造卵器synascusとなることを明らかにした。そして、この有性生殖を確認したことから本菌を子嚢菌門の菌種Pericystis apisと提唱することを支持した。その後、SpltoirとOlive(1955)は本菌に関する詳細な真菌学的観察をおこなって本菌を再分類し、Ascosphaera apis(以下A. apis)と命名した。
　現在では、世界各地でチョーク病の発生が散発し、その対応策について種々研究されている。そして飼養改善、ハイブリッド等による抗病性の研究、女王蜂の選択、抗菌製剤の開発、抗真菌剤の合成、他疾病との合併症対策等も検討されつつあるが、決め手となる防除法はなかなか見つからないのが現状である。
　わが国では宇田川らがカナダ産の輸入蜜蜂および秋田県産蜜蜂から、また山崎が蜜蜂中の菌類分布について調査した際に鳥取県および秋田県下で採取した蜜蜂中から、それぞれA. apisを分離した報告があるが、蜜蜂チョーク病の発生報告は未だなかった。
　このように蜜蜂のチョーク病に関して、わが国における発生実態および病性経過所見等の掌握は必ずしも十分とは言い難い状態にあるが、本病は1997(平成9)年の「家畜伝染病予防法の改正」で新たに「届出伝染病」に追加指定されるに至った。蜜蜂の需要増加と相まって輸入品の増加ならびに検疫業務の必要性から本病の研究が望まれていた。
　著者は、家畜衛生の業務遂行上において本病の自然発生例に遭遇し、本病防遏のための衛生指針を策定する上で必要となる基礎的事項について研究する機会を得た。
　本論文は蜜蜂のチョーク病に関して次の5項目:1)本病の自然発生例所見、2)A. apisの分布状況、3)分離A. apisの病原性、4)A. apisに対する抗菌剤のin vitro殺菌効果、5)本病の野外防除法について検討・研究したものであり、ここにその概要を報告する。
1. 蜜蜂チョーク病の自然発生例について
　チョーク病の自然発生例は、1979年3月に九州から岐阜県内に移動してきた蜂群:100群中3群に異常な蜜蜂の蜂児が認められた。蜂児は白色および黒色暗褐色でミイラ化しており巣脾の中および巣門の入り口に多数認められた。女王蜂周辺には少数の働蜂しか見られず、蜂勢は弱体化していた。
　チョーク病の病理組織学的所見ではミイラ蜂児のクチクラ層の内にPAS染色で赤く染まる多数の真菌を認め、菌糸は棒状、有隔壁性でY字状に分岐した菌糸も存在し、子嚢果(胞子嚢)の形成も確認された。
　チョーク病の病原学的検査では、血液寒天培地、卵黄寒天培地にて細菌は分離されなかったが、真菌培地で培養3日目より白色綿毛状の真菌が発育し、その菌糸および子嚢果(胞子嚢)の形態学的所見からA. apisが推定された。
　分離真菌は糖要求性強く、M40Y寒天培地による1週間培養にて薄膜性の子嚢果(胞子嚢)、球状の子嚢(胞子球)、楕円形の子嚢胞子を造り形態学的に子嚢菌類に分類されるA. apisであると最終的に同定された。
　本症例はわが国初のチョーク病の自然発生例であるが、本症発生が確認された1979年前後の年は夏期に30℃以上の気温が全国的に続いたことも本真菌症の発生に関与したのではないかと推察された。
2. Ascosphaera apisの分布状況
　本菌の分布については養蜂家飼養中の蜜蜂蜂児、蜂蜜、花粉について検討した。その結果、蜂児では64戸中16戸(25.0%)の養蜂家の蜂群に白色ミイラ蜂児が認められ、A. apisは320郡中80群(25.0%)から分離された。ミイラ蜂児が確認された蜂群では一見健康と思われる蜂児からも本真菌が分離された。
　蜜蜂では64戸320群中10戸16群(5.0%)からA. apisが分離され、花粉では64戸中3戸(4.7%)にA. apis陽性であった。
3. Ascosphaera apisの病原性
　本菌の病原性は、健常蜂児に胞子液を噴霧して感染状況を観察した。病像の出現率は、胞子濃度が10^3、10^4、10^9/mL(50mL/巣脾片面)と濃くなるほど発病率は高く、それぞれ40.0%、55.0%、93.0%の陽性結果であった。
　蜂児の感受性は4～5日齢までが高いが、3日齢以前の幼虫は死亡融解、成蜂による除去のためチョーク病の発生は確認されなかった。
　人工感染試験での接種真菌の回収では白色ミイラ蜂児からA. apisがいずれもほぼ純培養状に検出された。
4. Ascosphaera apisに対する抗菌剤のin vitro殺菌効果
　A. apisに対する薬剤の殺菌効果は、各薬剤希釈液に本菌の胞子を20℃で2.5、5、10、15分間作用させた後、M40Y培地にて培養し真菌発育の有無で判定した。その結果、消毒剤では逆性石鹸で1,600倍希釈液15分間、両性石鹸では800倍希釈液10分間、ヨード剤では3,200倍希釈液15分間の処理で殺菌効果を認めた。
　防黴剤14種中、プロピオン酸ナトリウム(PNa)のMICは0.04～1.25mg/mLであり比較的低濃度であった。
　殺菌性ガスのホルムアルデヒド(FA)ガス(Formalin 40mL/m^3、22℃)、エチレンオキサイド(EO)ガス(10%、30℃)ともに0.5、1時間の作用時間では十分な効果を認めなかったが、24時間では100%の殺菌効果を示した。
5. チョーク病の防除法
　A. apisの人工感染(胞子10^9/mL、50mL/巣脾片面)3日後の巣脾に対する薬剤の殺菌・防除効果は、逆性石鹸800倍希釈液(100mL/感染巣脾片面)による噴霧で8日目にミイラ蜂児が完全になくなり十分な効果が認められた。また、0.5%PNa液(100mL/感染巣脾片面)の噴霧でも8日目には完全な防除効果を認めた。そして、この両薬剤試験巣脾を戻した巣箱における成蜂、蜂児、卵いずれにも薬剤の副作用は認められなかった。
　この試験蜜蜂群から毎日採取した蜂蜜における残留試験の結果では、逆性石鹸は3日目まで残留(1.4ppm)を認めたが、4日目以降では検出限界以下となったことから野外での応用が示唆された。また、PNaは1日目の残留量5,250ppmが2日目以降では検出限界(25ppm)以下となった。
　EOガスの殺菌効果は、A. apisの人工感染(胞子3×10^8/mL、10mL/巣脾片面、25℃48時間培養定着)後の巣脾につき検討した。その結果、100%殺菌効果は、作用温度20℃のもとEOガス濃度2～3%以上での6時間作用群に認められ、作用温度30℃ではガス濃度1～3%で6時間作用群、5%濃度では2時間作用群に確認された。
　自然感染巣脾に対する防除試験は、感染巣脾(wax)をサイズ9×15cmに切断して被験消毒液に24時間浸漬した後、M40Y寒天培地にて培養し真菌の生死を判定した。その結果、逆性石鹸の100倍、200倍、400倍、800倍希釈液では100%の殺菌・防除効果を認めたが、1,600倍希釈液は無効であった。ヨード剤では1,600倍希釈液でも防除効果が認められたが残香性強く実用不適と判断した。
　殺菌性ガスについては、FAガス(前掲法)での22℃・24時間暴露により88.9%(8/9)、EOガス(前掲法)での30℃・24時間暴露により100%(9/9)の効果が認められた。
　1979年3月、九州から岐阜県内へ移動してきた蜂群:100群中3群に白色や黒色のミイラ蜂児を認めた。病原学的検査では真菌用培地で白色綿毛状の糸状真菌を分離して2週間培養後に子嚢果、子嚢、子嚢胞子の形成が認められ、わが国で初めてチョーク病の発生を確認した。
　分離真菌を使って健康蜂郡に人工感染させることにも成功した。そして、本病防遇のための対応策について検討した結果、逆性石鹸、両性石鹸に防除効果を認め、防黴剤ではPNaが有望であった。また、空巣脾の消毒法としてはFAガス、EOガスによる殺菌が効果的であることが推定された。},
 title = {蜜蜂のチョーク病に関する研究},
 year = {}
}