Studies on the Prevention of Leucocytozoon Infection of the Chicken. : IV. The Ecological Findings of Culicoides arakawae.

The following researchs have been carried out on the behavior of Culicoides arakawae, the intermediate host of leucocytozoon infection of the chicken. From 1961 to 1964, C. arakawae have been collected by a light trap once or twice every week at the hen house of our laboratory and a farmer's hen house at Hataayu, Makiishi in Okayama, city. The following items were also investigated: 1.)the successive change of the number of C. arakawae flied into the hen house in the evening, 2.)the number of C. arakawae caught by an insect net at various heights, 3.)the successive change of the number of C. arakawae caught by a light trap placed in the hen house. The results obtained were as follows. 1. C. arakawae comes flying into the hen house when the mean temperature is higher than 10℃. But, when the mean temperature is higher than 10℃, there are no clear correlation between the temperature, humidity and weather and the number of C. arakawae caught by light trap. The sex ratio of C. arakawae caught by a light trap varies with temperature, and the higher the temperature is, the greater is the percentage of the female. The ratio of engorged females to the whole females also varies with temperature. When the temperature is lower than 20℃, the percentage of engorged females is almost always below 50. When the temperature is higher than that, the percentage is from 70 to 90. 2. The number of C. arakawae caught by a light trap placed in the hen house shows the normal distribution with the central axis at 2 am. 3. It is probably certain that C. arakawae does not hibernate in the imaginal stage in the neighbourhood of Okayama city. 4. The number of C. arakawae come flying into the hen house in the evening, is most numerous during one hour following sunset. And in the summer season, in the flies caught by a light trap placed in the hen house, the percentage of females is usually far greater than that of males, but the sex ratio of C. arakawae, come flying into the hen house, is about 1:1. 5. C. arakawae usually flies below 20 m on the ground

Studies on the Prevention of Leucocytozoon Infection of the Chicken. : V. The Leucocytozoon Infection in the Yearling Hen.

The investigations were carried out about the reinfection with leucocytozoon disease in yearling hens that were infected with this disease in the preceding year. From the investigations carried out in 1962, 1963 and 1964, the following results were obtained. 1. Among the 20 hens once infected with this parasite, the reinfection was not at all found in the same and the following years by the microscopic examination of blood smear sample made once every week. 2. Among the 22 yearling hens that were completely protected from leucocytozoon infection in the preceding year by the use of pyrimethamine or sulfadimethoxine, it was found by the blood examination that 11 hens (50%) were infected with this disease in the following year. 3. Eleven hens, in the blood of which no gametocyte had been detected in spite of the fact that they had been among the highly infected flock, were not at all infected in the following year. From the results mentioned above, we have come to the following conclusions. Hens recovered from natural infection of leucocytozoon disease acquire the strong immunity later. And the effect of pyrimethamine and sulfadimethoxine used in these experiments are quite perfect

Studies on the Prevention of Leucocytozoon Infection of the Chicken. : II. The Preventive Effect of Furazolidone on Leucocytozoon Infection of the Chicken.

In recent years, leucocytozoon infection of the chicken has spread almost all over this country, and has given the serious damage to the poultry industry. We have been testing various drugs for the prevention of this disease, and in this study we have tested the preventive effect of furazolidone (nf-180) with leucocytozoon infection of the chicken. The procedure and the results obtained are as follows. Day old 200 chicks(♂100, ♀100) of White Leghorn were devided into 5 groups. Every group contains 20 males and 20 females. Group I was given the diet free from furazolidone as control, group 2 was given the diet containing 0.0005 % of furazolidone, group 3 the diet containing 0.001 % of furazolidone, group 4 the diet containing 0.005 % of furazolidone and group 5 the diet containing 0.02 % of furazolidone respectively. The smear samples of blood of all birds were made once every week for the detection of gametocyte of Leucocytozoon caulleryi. Body weights were measured at the same time. The infectivity rates of group 1(control), group 2, group 3, group 4 and group 5 were 64.1 %, 66.7 %, 70.2 %, 79.5 % and 48.6 % respectively. The infectivity rate of group 5 was slightly lower than that of the other groups, but from the practical point of view, it seems that furazolidone is ineffective for the prevention of leucocytozoon infection of the chicken at such concentrations. There were no significant differences between the weight gains of these groups

Studies on the Prevention of Leucocytozoon Infection of the Chicken. : VI. The Observation of the Gametocytes of Leucocytozoon caulleryi in the Unstained Wet Blood Film.

Usually the diagnosis of the infected chickens with leucocytozoon disease has been made by finding gametocyte of Leucocytozoon caulleryi in the blood smear stained by Giemsa's or May-Grunwald's method. But, as the inner part of the gametocyte can be observed in the unstained wet film of blood, the feature of the gametocyte considerably differs from that of the fixed sample. Method: One drop of blood from an affected chicken is placed on a cover-glass. This is inverted on a slide and the preparation is rimmed with paraffin. Then it is examined with phase-contrast microscopy at 37℃. Results and considerations: In macrogametocyte, the nucleus is rather small, usually have a curved or twisted rod shape and is suspended in protoplasm. The protoplasm is rich and filled with the immense number of fine granules. But in an usual smear sample, the nucleus shrinks and is covered with the granules at the time of smearing, and yet the granules are stained in dark purple by Giemsa's method, and hence it is considered that the shape of the nucleus is obscured. In microgametocyte, the nucleus is globular or oval, rather large in size compared with the size of the gametocyte itself, and placed at the nearly central point of the cell. The granules of protoplasm are a little larger in size and less in number than that of the macrogametocyte, and as the most of the granules are attached around the nucleus, these look like a pearl necklace in an optical section. As the nucleus expands at the time of smearing, it extends on nearly the whole surface of gametocyte and the protoplasm is seen indistinctly around it. As the granules are attached to the nucleus, they scatter on the surface of the nucleus as it expands. Most of the macro- and microgametocyte are covered with the membrane of the host cell. These cell membranes contain semifluid thinner than the protoplasm of the parasite. Some of which contain the granules of various sizes and they present the vivid Brownian motion. But these granules cannot be stained with Giemsa's dye

Studies on the Prevention of Leucocytozoon Infection of the Chicken : III. The Preventive Effect of Diaveridine.

1.diaveridineを飼料に最高0.01%から最低0.0001%までの各種の濃度になるように添加して,これを連続投与して本病の感染予防試験を行なった.その結果0.01%ならびに0.005%添加区においては感染が認められなかったが0.0025%以下の添加濃度では感染鶏が高率に認められた.従って本剤の最低有効濃度は一応0.005%の附近にあると思われる.しかしながら1964年度は,全国的に本病の予防剤としてpyrimethamineが発売されたため,岡山県下でも本病の発生が少なく,岡山大学農学部構内の鶏舎でも本病の感染率が一般に低かった.すなわち第1区の無添加対照区の感染率は35.0%であり,同時に行なった他の試験でも,薬剤無添加区での感染率は20～30%が普通であって,第1試験でみられたような濃厚感染は認められなかった.第2試験の0.001%添加区で81.3%の感染率を示したのはむしろ例外的現象と考えられる.従って第2試験の0.005%添加区がもしさらに濃厚な感染に曝された場合,果して完全に本病の感染を防ぎ得るか否かは疑問であるので,本剤を実地に応用する場合,最低有効濃度は0.005%より上にあると考えた方が安全と思われる.2.試験期間中における試験鶏の体重,産卵率,卵重等の推移を各区別の平均値で図示したように,いずれの場合も本剤がニワトリに対して毒性を示しているとは認められなかった.ただし第1図で,8月1日以後第3区および第4区の平均体重曲線が降下しているのは,第3区で2羽第4区で1羽がそれぞれ内臓型淋巴腫症に罹患し,体重が900gないし660gに減少したものが出たためであるが,8月29日現在の体重で,無添加対照区との間でF検定を行なっても5%の水準において有意の差は認められていない.第2図の平均産卵率の曲線についても,同様な傾向が見られるが,これも前記と同じ理由によるものと考えられ,期間中の総産卵個数について,対照区と各区の間でF検定を行なったが,いずれの場合も有意の差は認められなかった.3.ニワトリのcoccidium症に対するdiaveridineの効果は,前記の通りM. L. Clark3)によれば,sulfa剤と混じた場合0.001%であるといい,同じくpyrimethamineの効果は,S. B. Kendall&L. P. Joyner5)によると,sulfa剤と混合した場合0.004%が適当と認めている.従って,この両者はニワトリのcoccidiim症に対してはほぽ同等の効力を有しているものと見られるが,ニワトリのleucocytozoon病に対するpyrimethamineの最低有効濃度は著者ら2)の成績では0.00005%であるのに反し,diaveridineは前記の通り0.005%であるとすると,本病の予防効果では両者の間に100倍の開きが認められるわけであって興味深いことである.4.以上の試験により本剤単味の場合の最低有効濃度がほぼ0.005%であると判明したが,この濃度では実際の野外での応用の場合薬価の点で問題を生ずる恐れがある.したがってsulfa剤と混合投与した場合その相乗効果によりさらに投与濃度をさげる研究が行なわれないと本剤単味では応用範囲が限定されると考えられる

Studies on the Prevention of Leucocytozoon Infection of the Chicken. : I. The Behaviour of Culicoides arakawae to an Insecticide, DA-14-7.

In resent years, leucocytozoon infection of the chicken was found all over Japan except in Hokkaido. And it was revealed by AKIBA (1960) that Culicoides arakawae is the intermediate host of Leucocytozoon caulleryi. But the effective insecticide for C. arakawae is not yet discovered. In this study we have tested an insecticide named DA-14-7. This insecticide was manufactured recently for trial by a pharmaceutical company. The procedure and the results obtained are as follows. Two hen-houses of nearly the same scale were used for this experiment. In the control hen-house no insecticide was sprayed. In the experimental hen-house, emulsion of DA-14-7(diluted 1/20 with water) was sprayed on the ceiling, the wall, the beam, the pillar, the droppings board and other parts except the feeder and watering installations, at the rate of 300 ml. per m2. Oily solution of DA-14-7 was also sprayed on the hen body at the rate of 7 ml. per hen. These sprayings were repeated 5 times. The first spraying was carried out on September the 14th 1961. The second one on the next day. After the second spraying, the third, the fourth and the fifth spraying were carried out every four days. After spraying, the number of C. arakawae caught by light trap in the experimental hen house did not decrease, but the ratios of engorged female flies markedly declined. The ratios of engorged C, arakawae caught in the control hen house and the experimental hen house before spraying were between 83 % and 97 %. Seven hours after spraying, the ratios of engorged flies caught in the experimental hen house were from 6.3 % to 1.1 %, and 24 hours later 18.8 %, and 72 hours later from 34.4 % to 38.4 %. We cannot be satisfied with the effect of DA-14-7 as a repellent for C, arakawae. But, if the effective period of sprayed DA-14-7 is made longer and sprayed in relatively short intervals, the DA-14-7 is effective for protecting the chicken from suffering with C. arakawae, and consequently, the opportunity of the chicken to contact leucocytozoon infection and the degree of infection may be diminished considerably

Studies on the Prevention of Leucocytozoon Infection of the Chicken : VII. Preventive Effects of Pyrimethamine and Sulfadimethoxine Medicated Singly or in Combination

ニワトリのleucocytozoon病は,わが国の養鶏界に大きな損害をあたえた.秋葉ら1),2)が1958年に,本病の病原体として,Akiba(=Leucocytozoon) caulleryiの同定をおこなって以来,多くの研究者3,4,10～14,22～24)によって,この病原虫に有効な化学療法剤を発見しようとする色々の試みがなされた. 秋葉ら(3,4)は,Pyrimethamineとある種のsulfa剤が本虫に有効であることを示唆した.しかしPyrimethamineを0.004ないし0.005%飼料中に添加した場合は高い毒性を示した.一方,Lux20)(1954)がPyrimethamineとある種のsulfa剤をEimeria tenellaの予防に便用した場合認められる相乗効果について報告して以来,Plasmodium gallinaceumについてはRollo21)(1955)により,toxoplasmosisについては,Eyles&Coleman7)(1955),Eyles&Frances8)(1955),Frenkel&Hithings9)(1957)らにより,E. tenella感染症については,Joyner&Kendall15)(1955),Kendall&Joyner16,17,18)(1956～1958)およびBall5)(1960)らによって多くの研究がおこなわれた.そこで,本研究が,ニワトリのleucocytozoon病を予防するための,pyrimethamineとsulfadimethoxineの実際的な投与方法を発見するためにおこなわれた.実験はまず,両薬剤の低濃度における単味投与についておこない,ついで,A. caulleryiの感染に対し,これらの薬剤を混合投与した場合に相乗効果があるならば,これを明らかにしようとすることを試みた.飼料中に単味または,混合して添加した薬剤の効果は,対照区および試験区について,それらが本病原虫の自然感染に,さらされた後の感染率を比較することにより,検査した.1.単味添加したpyrimethamineの予防効果表2,に示すようにpyrimethamineは,0.0001%または0.00005%というような低濃度を添加した場合でも,本病の感染を完全に予防しえたが,0.00001%添加では予防しえなかった.(Exp.I and II)この結果は,Exp.IIIによって再び確認され,また,0.000025%の濃度に添加した場合は無効であることがわかった.上述の結果から,本病からニワトリを完全に予防するための,pyrimethamineの最低有効濃度は,0.00005%であると考えられた.2.単味投与した場合のsulfadimethoxineの予防効果単味投与したsulfadimethoxineの予防効果は,表3に示すようにして試験された.これらの結果から,本剤の最低有効濃度は,0.0025%であって,0.001%では無効と判定された.3.混合投与した場合のpyrimethamineとsulfadimethoxineの予防効果表4,に示したようにpyrimethamineとsulfadimethoxineを混合投与した場合は,それぞれ0.00001%および0.00025%を使用した場合でも完全に本病を予防しえた.すなわち混合投与した場合は,pyrimethamine1/5量に,sulfadimethoxine量を1/10量に減じうることが判明した.4.DA-106の予防効果抗コクシジウム剤として,一製薬会社により試作されたDA-106を使用しての試験Exp.VIIにより,上述の結果が確認された.0.008%の割合に添加されたDA-106は,本病に対して,完全予防効果を示した.この場合,飼料中のpyrimethamineとsulfonamide(sulfadimethoxineとsulfaquinoxalineの両方)の濃度は,それぞれ,0.000016%および0.00024%であった.これら一連の試験の結果をグラフ上に点描すると,図1の通りである.本図はpyrimethamineとsulfadimethoxineがA. caulleryiに対する効果において著明な相乗効果を有することの明らかな証拠を示している.これらの混合した薬剤の,無効な濃度の詳細がえられなかったので,限界曲線(点線)は仮想的なものにすぎない.飼料中に添加した0.0001%のpyrimethamineが広く使われるようになったため,わが国における本病は,ほとんど,制圧されてしまったので,本病の自然感染に依存しているような試験は続けることが困難となっている.しかしながら,pyrimethamineとsulfadimethoxineの混合投与方法を野外でおこなうことは安全性と経済性の観点から著明な改良をもたらすものと考えられる

Studies on the Prevention of Leucocytozoon Infection of the Chicken : VII. Preventive Effects of Pyrimethamine and Sulfadimethoxine Medicated Singly or in Combination

ニワトリのleucocytozoon病は,わが国の養鶏界に大きな損害をあたえた.秋葉ら1),2)が1958年に,本病の病原体として,Akiba(=Leucocytozoon) caulleryiの同定をおこなって以来,多くの研究者3,4,10～14,22～24)によって,この病原虫に有効な化学療法剤を発見しようとする色々の試みがなされた. 秋葉ら(3,4)は,Pyrimethamineとある種のsulfa剤が本虫に有効であることを示唆した.しかしPyrimethamineを0.004ないし0.005%飼料中に添加した場合は高い毒性を示した.一方,Lux20)(1954)がPyrimethamineとある種のsulfa剤をEimeria tenellaの予防に便用した場合認められる相乗効果について報告して以来,Plasmodium gallinaceumについてはRollo21)(1955)により,toxoplasmosisについては,Eyles&Coleman7)(1955),Eyles&Frances8)(1955),Frenkel&Hithings9)(1957)らにより,E. tenella感染症については,Joyner&Kendall15)(1955),Kendall&Joyner16,17,18)(1956～1958)およびBall5)(1960)らによって多くの研究がおこなわれた.そこで,本研究が,ニワトリのleucocytozoon病を予防するための,pyrimethamineとsulfadimethoxineの実際的な投与方法を発見するためにおこなわれた.実験はまず,両薬剤の低濃度における単味投与についておこない,ついで,A. caulleryiの感染に対し,これらの薬剤を混合投与した場合に相乗効果があるならば,これを明らかにしようとすることを試みた.飼料中に単味または,混合して添加した薬剤の効果は,対照区および試験区について,それらが本病原虫の自然感染に,さらされた後の感染率を比較することにより,検査した.1.単味添加したpyrimethamineの予防効果表2,に示すようにpyrimethamineは,0.0001%または0.00005%というような低濃度を添加した場合でも,本病の感染を完全に予防しえたが,0.00001%添加では予防しえなかった.(Exp.I and II)この結果は,Exp.IIIによって再び確認され,また,0.000025%の濃度に添加した場合は無効であることがわかった.上述の結果から,本病からニワトリを完全に予防するための,pyrimethamineの最低有効濃度は,0.00005%であると考えられた.2.単味投与した場合のsulfadimethoxineの予防効果単味投与したsulfadimethoxineの予防効果は,表3に示すようにして試験された.これらの結果から,本剤の最低有効濃度は,0.0025%であって,0.001%では無効と判定された.3.混合投与した場合のpyrimethamineとsulfadimethoxineの予防効果表4,に示したようにpyrimethamineとsulfadimethoxineを混合投与した場合は,それぞれ0.00001%および0.00025%を使用した場合でも完全に本病を予防しえた.すなわち混合投与した場合は,pyrimethamine1/5量に,sulfadimethoxine量を1/10量に減じうることが判明した.4.DA-106の予防効果抗コクシジウム剤として,一製薬会社により試作されたDA-106を使用しての試験Exp.VIIにより,上述の結果が確認された.0.008%の割合に添加されたDA-106は,本病に対して,完全予防効果を示した.この場合,飼料中のpyrimethamineとsulfonamide(sulfadimethoxineとsulfaquinoxalineの両方)の濃度は,それぞれ,0.000016%および0.00024%であった.これら一連の試験の結果をグラフ上に点描すると,図1の通りである.本図はpyrimethamineとsulfadimethoxineがA. caulleryiに対する効果において著明な相乗効果を有することの明らかな証拠を示している.これらの混合した薬剤の,無効な濃度の詳細がえられなかったので,限界曲線(点線)は仮想的なものにすぎない.飼料中に添加した0.0001%のpyrimethamineが広く使われるようになったため,わが国における本病は,ほとんど,制圧されてしまったので,本病の自然感染に依存しているような試験は続けることが困難となっている.しかしながら,pyrimethamineとsulfadimethoxineの混合投与方法を野外でおこなうことは安全性と経済性の観点から著明な改良をもたらすものと考えられる

PAM (2-pyridine Aldoxime Methiodide) Therapy and its Preventive Effect for Alkylphosphate Poisoning of Fowl.

Formerly, atropine was used as an antidote for alkylposphate poisoning. And in 1955 I. B. WILSON suggested PAM (2-pyridine aldoxime methiodide) as a specific antidote for alkylphosphate poisoning. In 1956 K. HIRAKI et al. introduced PAM in Japan. Since that time, the PAM therapy for alkylphosphate poisoning has been widely practised in this country. In recent years, as the fowls were frequently subjected to alkylphosphate poisoning in Okayama district, we had investigated therapeutic and preventive effects of PAM for alkylphosphate poisoning of fowl. The alkylphosphate used in this experiment was parathion (diethyl p-nitrophenyl thiophosphate) and the method we employed for determining the cholinesterase activity of serum was MICHEL's method. Hens and chickens of White Leghorn, Barred Plymouth Rock and Rock Horn were used in this experiment. The results we obtained were as follows. 1. The lethal doses of parathion for fowl were 4-6 mg/kg in chichen and 10 mg/kg in hen. 2. With intravenous injection of 100 mg/kg of PAM, within 20 minutes after oral LD of parathion, conditions of fowls remarkably recovered compared with controls. The administrations of PAM of same quantity were more effective when they were divided into two or three doses. Alkylphosphate poisoning caused no retained disturbance sin recovered chickens of which ovary not yet developed. 3. In the case of laying hens, even if PAM administration prevents them from death, they lose their economical value. Because, PAM cannot prevent hemorrhage in the ovary and ruptured yolk caused by the poisoning, and so poisoned hens cease the egg laying for following two or three months. 4. As a effective period of time of intravenously injected PAM is only one hour or so, we cannot expect too much of the preventive effect of PAM for alkylphosphate poisoning of fowl in the fields