@misc{oai:az.repo.nii.ac.jp:00003246,
 author = {須永, 藤子 and Sunaga, Fujiko},
 month = {2013-06-29, 2014-08-19},
 note = {Babesia infection of dogs is caused by B. gibsoni, which infects animals belonging to Canidae in the erythrocyte and is transmitted by ticks, and develops pyrexia, splenomegaly and severe hemolytic anemia. This protozoan was first detected by Patton in 1910, since then the infection was frequently reported from different areas of Asia. In Japan, it is widely distributed mainly in the western part and recently, the infected area is spread farther to the eastern part. Hitherto, an immediately effective drug, Ganaseg, a diminazen preparation, was used for treatment, but its manufacture was discontinued because an accident occurred in the manufacturing process. Since then, the product was not obtained in good quality in spite of the improvement of the process, while the fatal cases of dogs treated with the product were increased. Consequently, the development of protective vaccines is earnestly desired.
 In babesiosis, death is caused in the process that the sporozoites injected into dogs by infected ticks invade the erythrocytes, in which the parasites developed to erythrocytic merozoites, and then they multiply by asexual reproduction and lead the blood cells to destruction and resultant anemia of dogs. Therefore, an experiment was attempted to examine the protective efficacy of the proteins originating from the merozoites or attenuated merozoites. First, in vitro culture method were examined to produce the steadily propagating isolate. Secondly, attenuation of the isolate was attempted by subculture and the resultant isolate was examined for immunogenicity. Thirdly, inactivated vaccines with more safety were examined by two antigens; β - propiolactone-treated merozoite antigen and exoantigen derived from the culture supernatant of merozoite in vitro culture.
 The results of the present study are summarized as follows.

1. Production of the merozoite isolate by in vitro culture
 Although the life cycle of B. gibsoni is still partially indistinct, the sporozoites injected to dogs by infected ticks immediately invade the erythrocytes and multiply by binary fission, absorbing necessary nutritive elements and microelements from the blood. Many merozoites multiplied are released from the broken erythrocytes into the plasma and then they attack uninfected cells. The factors necessary for culture of merozoites are insufficiently elucidated because the parasite has such complicated cycle as described above. However, it is known from the results obtained from the in vitro culture of bovine Babesia species that serum is necessary for culture of the parasite and its quality differs delicately in different Babesia species. Therefore, the concentration of serum added to the liquid culture medium, RPHI-1640 (GIBCO), was examined and the result obtained was that merozoites developed insufficiently under a concentration of 10%, while their development was inhibited in a concentration more than 50 %. So, in the following experiments, infected erythrocytes were cultured in the RPMI1640 liquid medium supplemented with 20% of serum.
 Infected canine erythrocytes was diluted with the culture medium to a PCV of 8-12%. The suspension, 2ml each, was divided in 12 wells of culture plate. The plate was incubated at 37℃ in a humidified atmosphere containing 5% CO_2. In each well, 1ml of the overlying medium was removed and replaced with fresh medium every day, at the same time, Giemsa-stained blood smears were made to determine parasitemia. A subculture was usually started after 3 days incubation diluting the old culture by 1:3 with a fresh suspension of normal erythrocytes.
 The merozoites in erythrocytes were not well developed from the 2^nd to 28^th subculture. So, the period of culture was prolonged to 15days. On Days 3, 6, 9, 12 and 15 of the period, some of the cultures were tentatively subcultured to examine the multiplication of merozoites. The procedure used was as follows: a part of infected erythrocytes were removed from a well to another, which a proper volume of fresh suspension of canine erythrocytes was added to and the mixture was cultured for 15 days. From the cultures described above, the culture containing the most multiplied merozoites was selected and further subcultured. Consequently, from the 23^rd subculture, the merozoites rapidly proliferated from day 1 of culture, and from the 29^th subculture, the merozoites showed a peak of multiplication from day 2 to 4. After then, subculture was carried out every 3 days. As a result, parasitemia, denoted by a percent of infected erythrocytes in 1000 erythrocytes measured, reached 3.1±1.2%. Thereafter, the rate increased and the isolate showing a parasitemia of 18.8 ± 1.3 % was established at the 190^th subculture.

2. Production of the attenuated merozoite isolate and its protective effect of the challenge infection by the virulent isolate
 The merozoite isolate was established as described above. Next, the attenuated isolate should be made, so the period necessary for attenuation of the isolate was confirmed and further the protective effect of the attenuated isolate was examined by challenging the dogs pre-inoculated with the attenuated isolate.
 The attenuated isolate was evaluated with the indexes of hemolytic anemia and pyrexia. First, one female beagle, one-year old, was inoculated with 10^9 merozoites of the 200^th subculture and the pathogenicity of merozoite was observed in dogs for 60 days. The 200-subcultured isolate caused no pyrexia but a slight anemia with a PCV value of 30%. Parasitemia reached 4.4% of erythrocytes on day 32 after inoculation. The subnormal value of PCV indicated that the isolate was insufficiently attenuated.
 Two one-year old female beagles inoculated with the 400^th subcultured merozoites, however, developed no pyrexia nor was recognized anemia, a PCV value being above 40%. No change was also seen in appetite and health. Parasitemia was retained under 0.01% throughout the experimental period, exceptionally 0.4% in dog 2 on the 38^th day after challenge, and yet parasites appeared sporadically in the peripheral blood. These results showed that the 400^th subcultured isolate was sufficiently attenuated. The isolate was examined for immunogenicity by the indirect fluorescence and Western blot 60 days after inoculation, and the antibody titer showed 20,480 by the indirect fluorescence. Furthermore, the main seven bands from 22 to 127 kDa were observed with both antiserum of dogs inoculated with the 400^th subcultured merozoites and recovered from challenge infection with the virulent isolate by the Western blot. These results clearly indicated that the 400^th subcultured merozoite isolate had the same degree of immunogenicity as of the virulent isolate.
 In the next step, the 400^th subcultured isolate or attenuated merozoite isolate was examined for the activity protecting the attack by disease. Two female beagles, one year old each, were inoculated with the attenuated isolate and the protective activity of the isolate was examined using such indexes as the suppression of the multiplication of parasite and anemia indicated by PCV and the reduction of pyrexia, after challenge infection with the virulent isolate. In a result, the group pre-inoculated with the attenuated isolate showed the singular effect suppressing the multiplication of parasite and anemia. Further, the isolate had marked activity reducing clinical signs, especially pyrexia, because the treated group of two one-year old beagles showed no pyrexia although the control group suffered from pyrexia for 8 days. These results indicated that the attenuated stain met such qualifications of live vaccine as non-pathogenicity and effective immunogenicity, furthermore, suppressive effect of the multiplication of parasite and reductive activity of clinical signs such as anemia and pyrexia after challenge infection by the virulent isolate.
 However, the attenuated isolate will be limited to apply as live vaccine with because the parasite inoculated will survive in the dogs, which become carriers, so the application of the isolate as live vaccine will be limited to the endemic area.

3. The effect protecting the attack of disease by the inactivated antigens: the culture supernatant antigen and β propiolactone treated merozoite antigen
 The attenuated isolate consisting of steadily propagating merozoites was established and examined to reveal that it showed no side effect but marked effect protecting the attack by disease. The isolate, however, should be limitedly applied as live vaccine, so the inactivated vaccines with more safety were examined.
 Two kinds of antigens were examined as immunogen. The supernatant antigen was made by concentrating the supernatant of culture medium with the ultrafilter and the merozoite antigen by treating the merozoites, which were obtained by hemolyzing the cultured infected erythrocytes, with β propiolactone. These immunogens were subcutaneously injected to two female beagles one-year old. Both antigens were examined for immunogenicity by the indirect fluorescent method and Western blot 68 days after inoculation. The result obtained was that the antibody showed a titer of 2,560-10,240 and showed the same seven main bands from 22 to 128kDa as recognized by Western blot in the serum of dogs recovered from challenge infection with the virulent isolate. This result indicated that both inactivated antigens had the like immunogenicity as of the virulent isolate. Then, two groups of dogs inoculated with both antigens, respectively were challenged with the virulent isolate and both inactivated antigens were compared to each other on the preventive activity of the attack with such indexes as the preventive rate of the multiplication of parasites, anemia denoted by PCV value and pyrexia. Consequently, the marked suppressive effects of the multiplication of parasites and pyrexia were recognized in both group of dogs, especially the suppressive effect of anemia was excellently observed in the group inoculated with the supernatant antigen. Further, this antigen could be manufactured in large quantities and easily refined as vaccine.
 The above results clearly showed that the antigenic substance recognized in the supernatant of culture medium had an excellent activity as inactivated vaccine.

 The present study clarified that the steadily proliferating isolate of Babesia gibsoni was successfully established by in vitro culture and the 400^th subcultured attenuated isolate was effectively used as live vaccine, and further, the supernatant antigen and β-propiolactone treated merozoite antigen had an excellent effect suppressing the attack of babesiosis. The comparison of these inactivated vaccines about the suppressive activity of anemia and safety led the result that the supernatant antigen of culture medium was the more usefulness., Babesia gibsoni症は、イヌ科動物に感染性を持つBabesia gibsoniがマダニの媒介によってイヌ赤血球に感染し、発熱、脾腫、強度の溶血性貧血を起こす原虫性の疾病である。本原虫はPatton(1910)によって初めて検出されて以来アジアでの報告が多く、わが国では西日本を中心に分布し、近年、その汚染地域が東日本にも拡大しつつある。従来、本症例の治療には即効性のガナゼック(ジミナジン製剤)が用いられたが、本剤は製造工程で問題が生じ、製造中止となったため、イヌ重症例での死亡率が増加しており、ワクチンの開発が望まれている。
　B.gibsoni症でイヌが死亡する原因は、汚染マダニの吸血と共に注入されたスポロゾイト虫体がイヌ赤血球内に侵入して赤内型メロゾイトとなり、メロゾイトが無性生殖によって異常に増殖し、急激に大量のイヌ赤血球が破壊され、イヌに極度の溶血性貧血が発生することによる。そこで、メロゾイトの異常増殖を抑制し、発熱、貧血などの発症を軽減する効果が弱毒化メロゾイト株とその不活化抗原に存在するかを明らかにする実験を行った。
　弱毒化メロゾイト株とその不活化抗原を得るために、メロゾイトの培養方法を検討し、安定的に増殖する培養メロゾイト株の作出を試みた。次いで、この培養メロゾイト株の弱毒化を試み、その弱毒化したメロゾイト株の発症防御効果について検討した。さらに、安全性の高い不活化ワクチンを開発するためにメロゾイトの培養上清中に認められた可溶性の抗原と培養感染赤血球から採取してβ-プロピオラクトンで処理した不活化メロゾイト抗原の発症防御効果について検討した。
　本論文の概要は次の通りである。

1. B.gibsoniメロゾイト培養株の作出
　B.gibsoniの発育環は不明な部分もあるが、マダニにより感染したスポロゾイトは直ちにイヌ赤血球に侵入し、必要な栄養素や微量成分を血液から得て2分裂を繰り返し増殖する。増殖した多数のメロゾイトは赤血球を破壊して血漿中に遊出し、新たな赤血球に侵入し、再び増殖する。このようなサイクルで増殖を繰り返すメロゾイトを培養器内で増殖させる場合、増殖に必要な因子の解明はまだ不十分である。しかし、牛バベシアのメロゾイトの人工培養の結果から、培養には栄養となる血清が必要で、その量は原虫の種によって微妙に異なることが知られている。そこで、メロゾイトの培養に用いる液体培地(RPMI1640)に栄養分として添加するイヌ血清濃度を検討した結果、イヌ血清の添加が10%以下では原虫の発育が悪く、50%以上添加すると返ってメロゾイトの増殖が阻害されることが明らかとなったので、以下の実験ではRPMI1640液体培地にイヌ血清を20%の割合に添加して感染赤血球を培養した。
　メロゾイト感染赤血球の培養方法はメロゾイト感染赤血球1容に培養液を9容加え、これを12ウエルの平底プレートに2ml(0.53ml/cm^2)ずつ分注して、5%CO_2/air、37℃下で培養した。各ウエルの培養液は毎日、1mlの培養上清を新鮮な培養液と交換し、この時、ギムザ染色血液塗抹標本を作製し、parasitemia(赤血球1000個中のメロゾイト感染赤血球数を百分比で示したもの)を算定した。赤血球内に感染しているメロゾイトの継代方法はウエル内の培養感染赤血球液の1/3を別のウエルに取り、これに新鮮イヌ赤血球加培養液2/3を加えて混和し培養する方法を用いた。
　2代目から28代目までは、メロゾイトの赤血球内増殖が悪かったので、培養期間は15日間とし、継代は培養3日目、6日目、9日目、12日目および15日目にそれぞれ行なった。この中で最もメロゾイトの増殖が良いものを選び継代した結果、29代目からは培養3日目にメロゾイト増殖のピークが認められたので、以後の継代培養は3日目ごとに実施した。その結果、30代では培養3日目のparasitemiaは3.1±1.2%であったが、190代ではparasitemiaが18.8±1.3%を示すB.gibsoniメロゾイト培養株が作出できた。

2. 弱毒化メロゾイト株の作出および作出株の発症防御効果
　継代培養が可能な強毒メロゾイト株が作出できたので、この株を何代継代すれば弱毒化するかを検討し、この弱毒化メロゾイトの発症防御効果を検討した。
　何代継代すれば弱毒化するかの判断は、溶血性貧血と発熱を指標にして行った。まず、200代継代した培養メロゾイトの10^9個を1頭のビーグル犬(1歳、雌)に接種し、60日間にわたって貧血と発熱を指標に病原性を観察した結果、発熱は認められなかったが、軽度の貧血(PCV 30%)が認められた。parasitemiaは、接種後32日目に4.4%を示した。PCVが正常値より低いという結果から弱毒化が不十分であることが明らかとなった。
　同様に400代継代した培養メロゾイトの109個を接種した2頭のビーグル犬(1歳、雌)では発熱や貧血(PCV 40%以上)は認められず、元気・食欲にも変化はなかった。実験期間を通じてparasitemiaは0.01%以下(No.2に38日目のみに0.4%)であった。これらの結果から400代継代メロゾイトは、弱毒化されたと判断した。400代継代メロゾイトの免疫原性を間接蛍光抗体法とウエスタンブロット法で確認した結果、間接蛍光抗体法による血中抗体は20,480倍を示した。さらに、この血中抗体には、強毒メロゾイトで攻撃感染後、回復したイヌの血清中にウエスタンブロット法により確認された主要な7本のバンド(22kDaから127kDa)が認められた。以上の結果から400代継代メロゾイトは、強毒メロゾイト接種と同様のB.gibsoni抗体を誘導することが明らかとなった。
　そこで400代継代メロゾイト(弱毒化メロゾイト株)の発症防御効果を検討した。実験は弱毒化メロゾイトの10^9個を接種して免疫を獲得した2頭のビーグル犬(1歳、雌)を強毒メロゾイトで攻撃感染して、メロゾイトと貧血(PCV)の抑制度および発熱の有無を指標として発症防御効果を検討した。その結果、弱毒化メロゾイトで前処置した群は、強いメロゾイトの増殖抑制と貧血抑制効果を有することが明らかとなった。さらに、発熱から見た症状の軽減度は著しく、強毒メロゾイトを接種した対照群の2頭のビーグル犬(1歳、雌、)では発熱が8日間にわたって認められたが、免疫群では全く認められなかった。これらの成績から、この弱毒化メロゾイトは、生ワクチンとしての条件である病原性がないこと、免疫原性があること、メロゾイト増殖を抑制し、貧血や発熱などの臨床症状を軽減し、バベシア症の発症抑制効果を有することが明らかとなった。
　しかし、弱毒化メロゾイト株を生ワクチンとして野外で応用した場合には、免疫に用いた弱毒化メロゾイト株が生体に残存し、免疫犬がキャリアとなる危険性があるため、生ワクチンとしての使用は汚染地域に限定されよう。

3.不活化抗原(培養上清抗原・β-プロピオラクトン処理メロゾイト抗原)の発症防御効果
　安定的に増殖する弱毒化メロゾイト株を作出し、この株の生ワクチンとしての性質を検討した結果、この株には接種による副作用はなく、強い発症防御効果を示すことが明らかになった。しかし、生ワクチンとして使用する場合、接種メロゾイトが長期間にわたって血中に残存することが想定されるので、安全性の高い不活化抗原を作製し、その発症防御効果を検討した。
　不活化抗原はメロゾイト培養上清液を限外濾過法で濃縮した培養上清抗原と培養感染赤血球から採取してβ-プロピオラクトンで処理した不活化メロゾイト抗原の2種類を用いて、発症防御効果を検討した。実験はそれぞれの抗原にQuilAアジュバントを加え、それを20日間隔で3回、各2頭のビーグル犬(1歳、雌)に皮下投与し、初回投与後68日目に強毒メロゾイトで攻撃感染して、メロゾイトと貧血(PCV)の抑制度および発熱の有無を指標にして発症防御効果を検討した。両群ともに初回抗原投与後68日目の間接蛍光抗体法による血中抗体価は2,560-10,240倍を示した。これら免疫群に強毒メロゾイトで攻撃感染したところ、両群ともに強いメロゾイトの増殖抑制と発熱の防止効果が認められた。貧血抑制効果は両群ともに認められたが、培養上清抗原で免疫した群に強く認められた。
以上の事実から、培養上清抗原は強い発症防御効果を示し、安全性に優れ、大量生産が可能で、赤血球膜の混入がなく、精製が容易であることが明らかとなった。
　培養上清抗原(可溶性抗原)の性状を知るために、この抗原をイヌに接種し、初回免疫後68日目に得られた抗体をウエスタンブロット法で分析した結果、強毒メロゾイトを接種し回復した犬、および不活化メロゾイト抗原で免疫した犬の血清中に認められたメロゾイト抗原に対する主要な7本のバンド(22kDaから127kDa)と同じ位置にバンドが確認された。このことから、培養上清抗原には主要なメロゾイト抗原が含まれていると判断された。
　以上、効果および安全性の点からB.gibsoni培養上清中に認められる可溶性抗原物質が不活化ワクチンとして優れた作用を示すことが明らかとなった。

　本研究は、強毒メロゾイトから安定的に増殖するB.gibsoniメロゾイトを作出し、このメロゾイトを400代継代することで弱毒化メロゾイト株の作出に成功した。この弱毒化メロゾイト株、その培養上清抗原とβ-プロピオラクトン処理メロゾイト抗原にはバベシア症の発症を抑制するワクチン効果のあることを明らかにし、さらにワクチンとして実用化するには安全性の高い培養上清抗原を用いた不活化ワクチンが最も優れていることを明らかにした。},
 title = {Babesia gibsoniの弱毒化メロゾイト株の作出とワクチン効果に関する研究},
 year = {}
}