豚コレラウィルスの組織培養に関する研究 II. 培養組織による豚コレラウィルスの累代培養

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In place of the suspended cell culture method used bv most investizators in therast, ENDERS and his co-workers r>lasma-clot culture-method was used. Using thesvnthetic medium No. 199, containinz horse serum, roller tube culture of spleenand kidney tissues were undertaken.Hog cholera virus was first injected into and then cultivated in the tissue cultureand then transfered into another one. Tissue culture of the lVIiyazaki strain of 9rassaues and the ALD strain of 4 rassazes were infected into the swine. On the12th passage of the lVIiyazaki strain, infective fiter was also determined.From the results of clinical findings, percentage of leucocytes, anatomical viewsand bacteriological examination, it was confirmed that the pathogen which producedthe disease in all the pigs tested and which caused their deaths, was hog choleravirus descended from the one used for inoculation. The infective fiter was a dilu-lion of 10 It stood 10 days in Miyazaki strain and 17 days in ALD strain asa course of disease. This fact seemed strange, because it is characteristic that thecourse of the disease is longer in the Miyazaki strain than in the ALD strain whenfresh, natural blood virus is injected. If the prolongation of the course in the ALDstrain means the decrease of virulence, it may be considered that the Miyazakistrain is more suitable to the tissue culture than the ALD strain. In the groupof dilution 10- in the infeetive Liter test, febrile reaction appeared normal betweenthe 8th to 17th day of the disease and was supposed to recover and resist, but itwas followed by a secondary rising of fever and death on the 34 th to 37 th dayof the disease.It was uncertain whether the fact was caused as a result of the chronic course9of the disease owing to a dilute virus or whether it was caused by other, secondaryreasons. But, the characteristic changes indused by chronic hog cholera wererecognized. on autopsy, and the bacteriological examination was negative. Fromthese facts it was considered that the death was caused by hog cholera virus.Ferritin is an iron-containing protein which is stored maitnly in the marrowliver and spleen etc.. Above all, the richest source of ferritin is the reticulo-endothelial system of the horse spleen, but ferritin is not contained in the bloodof the jugular vein of the normal horse.On the other hand, it was found by means of a new complement fixationtest" that free ferritin was contained in the blood of the jugular vein of theanemia infected horse.Therefore, first of all, the proving for the new methed of the complementfixation test on the free ferritin in horse serum was tne result of this study.The results are summarized as follows :(1 ) This complement fixation test is a new method for the identificationof the antigen, that is, the free ferritin in the anemia infected horse serumthrough the use of the known rabbit antiserum for crystallizable horse spleenferritin as shown in Table 1.Especially, the antiserum was heated at 65 C for 30 minutes and the titerof 2 units was used. The antigen was prepared by the following method :At first horse serum was heated at 80C -85C for 5 -TO minutes. Then,the coagulum was stirred with a glass-rod and was then separated at 4000 r. p. m.for 20 minutes. The supernatant was used as the antigen.By this procedure the anticomplemental property, the non-specific comple-mental fixative property, and the suppressible property to the reaction werenearly removed and the specific antigen, that is, the free ferritin in the horseserum was extracted. -(Cf. Table 6).(2 ) The result of the complement fixation test with the use of the antigenextracted from an infectious anemia horse serum and the known rabbit antiserumis shown by the two dimension method in table 3. It is recognized that thepattern of the complement fixation test on the horse serum antigen is surroundedby a parabola as well as the result of the reaction on the crystalline horsespleen ferritin that is shown in table 2.Especially, in the reaction to the antigen of high density, a prozone pheno-menon caused by the excess of antigen is observed.(3 ) The complement fixation tests in the cases of most of the seraantigens of horses affected with equine infectious anemia were positive to theantigen dilution ranging frorn 1 : 1 to 1 : 15 but the precipitin tests werenegative, as is shown in Table 5.The complement fixation test on the serum antigen in the case of feverparoxysm in the seriously anemia infected horse, was positive in the antigendilution ranging from 1 : 40 to 1 : 250, and the precipitin test was, also, positivewith such serum.The Complement fixation tests on the sera antigens of healthy horses thathad been normal, histopathologically, were negative.(4 ) The antigens in infectious anemia horse sera were centrifuged andcondensed at 42000 r. p. m. -110000 g for 60 minutes. After the centrifugation,each antigen titer of the bottom sera in the complement fixation tests was inte-nsified from about 5 to 30 times more than each antigen titer of the originalsera, as shown in Table 7 and the reactions on the supernatant antigens ca-me to be negative.(5 ) The minimum positive density of the crystallin horse spleen ferritinin the complement fixation test was about 0.025 pg N. per ml, and in theprecipi
社団法人日本獣医学会の論文
1957-08-30

豚コレラウィルスの組織培養に関する研究 II. 培養組織による豚コレラウィルスの累代培養

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