levels of the spinal cord, clinical symptoms and cerebrospinal lesions were ... the vertebral column being bent, a short syringe needle was inserted through the median ..... A marginal space was clearly demonstrated around some organisms.
「
Mcd.1 『 Japan. J. Exp. LVol.42,3,p.2(3282,1972』
Neuropathology
of Intraspinal Infection
in Mice with the Tyzzer's Organism Takashi ONODERA and K6saku FUJIWARA
γ ′ ′P″力 ο gノ ,LS″ ″ι Dψ α ″2′ グИη れα ′ ο ιグ■イ ″θ α ′乱ル″ ″∫ ″ ι ″,υ″υ クグ 需ο りο “ P・ 0・ 7α た α ″ α ″α θ θ ″ ,1乃 秒ο′ ,Jψ α ,
(Rcceivedお r PubHcation,Ⅳ [arch 6,1972)
Surnrnary: After inoculation of infective liver
homogenate into mice at different levels of the spinal cord, clinical symptoms and cerebrospinal lesions were observed in relation to the propagation of organisms. Although fatal encephalitis was prevaiing after infection with a large dose, ascending flaccid paralysis probably resulting from the damage of anterior horn cells appeared in mice which had survived for a felv days after infection with a smaller dose. Spinal cord lesions were characterized by wide-spread necrotizing area with leucocyte infiltration in association with meningitis. Many organisms were present within the cytoplasm of nerve cells, sometimes also within the neurites or nuclei. Electron microscopy revealed that most organisms having peritrichous flagella rt ere located in parallel within the matrix of the nerve cell cytoplasm, as in hepatocytes, being isolated from one another. Some of them were shown within the cytoplasm of ependymal or glia cells as well as macrophages. In the brain and spinal cord of mice having survived after inoculation with smaller dose was shown a repairing process characterized by the disappearance of organisms and the proliferation of glia cells as well as reticular fibrils. The histopathology of brain lesions were essentially not different from that after inoculation into the cisterna cerebellomedullaris. INrnoouc'rroN
Naturally occurring Tyzzer's disease of mice is characterized by focal necrosis in the liver' hemorrhagic enterocolitis and rod-shaped organisms r,r,ithin the cytoplasm of hepatocvtes or enteric mucosal epithelial cells [2, 9, 25]. On the other hand, an encephalopathy was observed in mice infected intracerebrally or by the route of the cisterna cerebellomedullaris (c.c.m.). The presence of organisms within the nerve cells, glia cells, epithelial cells of the choroid plexus or ependyma'was demonstrated [8, 17, 1B]. While histo- and cytopathology and pathogenesis in the infection of hepatocytes and enteric epithelial cells have been studied extensively in mice [5, B, 9, 18, 2+,25] and rats U3, 27), neuro-cytopathology has not been fully studied. In our previous studies [8, 17], some interesting findings were obtained of the formation of brain lesions due to the propagation of organisms. Then, it has been considered that many large neurons existing in the spinal cord may be also target cells of which infection provides more informations on the neuro-cytopathology This. paper deals with cyto- and histo-pathological findings on the spinal cord and the brain of mice having been inoculated with infective materials intraspinally, with special reference to the relation between the multiplication of organisms and the functional disorders in the host animals. 小野寺
節 ,藤 原公策 (東 京 大学医科学研究所獣医学 研究部 )
264
T. ONODERA & K.
FUJTWARA
lYor,.42.
Marpnrar,s AND MB'rHoos ,lnirnals : ICR female mice of age 20 to 25 weeks weighing 30-40 g were obtained from Central Laboratory for Experimental Animals, Tokyo. They were fed commercial pellets (I\,[NF, Oriental Yeast Co., Tokyo) and water ad libitum. The breeding colony was checked {or Tyzzer's disease by serology with retired breeder mice [4] and also by cortisone injection test with young mice [10, 23]. Infectiae materials: The SK strain of organisms [6] being maintained by 300 or more passages in mice was injected intravenously into ICR mice which received 2.5 mg cortisoneacetate (Corton, Merck-Banyu Co., Tokyo) shortly after inoculation. A few days later, the livers showing multifocal necrosis were harvested and homogenized in phosphate buffer saline pH 7.2 (PBS). The number of organisms contained in the homogenate was estimated by a method described previously [4], and an appropriate dilution in PBS was used as infective materials. Inoculation.' Mice were anesthetized with ether and an incision I cm long was made on the dorsal skin across the vertebral column to expose subcutaneous tissues. Then, the vertebral column being bent, a short syringe needle was inserted through the median Iine of an objective intervertebral foramen, and 0.02 ml of infective liver homogenate was injected. Successful intraspinal injection was confirmed by u momental flaccid reaction in the hind paws observed immediately after injection. After inoculation animals were observed for 20 days, and clinical signs were recorded as described previously [I7]. Some animals were subjected to bacteriological and histopathological examinations. Vital staining.' According to Levine [4, 15], I ml of 0,5o/o aqueous solution of trypan blue was injected subcutaneously 12 hours before killing. Histopathological examination: The brains and spinal cords were fixed with l0o/o buffered formalin (pH 7.2), Carnoy solution or l0o/o formalin containing 2o/o acetic acid, and they were embedded in paraffin. Sections 6 to B p- thick were stained with hematoxylin and eosin, luxol fast blue-cresyl violet, or Watanabe's silver nitrate impregnation for reticulum fibrils [26], protagol-S method for neural axon [3]. Cajal's gold-sublimate method for astrocyte [6] or Gomori's methenamine silver nitrate method [12] was also applied. For the most definite staining of organisms in tissues, the methenamine method was modified and sections were treated with 5o/o chromic acid for 90 minutes and were impregnated at 60'C for 1 to 1.5 hr. Immunofluorescence tecltnique: Direct fluorescence antibody technique was applied to cold aceton-fixed sections for the detection of organisms in tissues. Fluorescein-labelled antibody was prepared from immune rabbit serum against Tyzzer-infected mouse liver, as described previously [7, I l]. In order to detect extravascular 1-globulin diffusion indirect technique was applied to cold aceton(-20'C)-fixed sections []. Phase contrast and electron microscop2.' The spinal cord was removed immediately after killing and fixed at 4"C for 6 hr in lo/o osmium tetroxide in veronal buffer at pH 7.4. The specimens were then dehydrated through graded concentrations of ethyl alcohol, and they were placed in a propylene oxide bath and embedded in Epon 812 resin. One g,m-sections were made from these specimens and examined by a phase contrast microscope. Then, the tissue blocks, in which many intracellular organisrns had been obsered, were subjected to electron microscopic studies. Ultrathin sections were made by a Porter-blum ultramicrotome, and were stained with lead hydroxide [20] and uranyl acetate 122). Observation was made using JEM-7 or Hitachi l1-A electron microscope.
INTRASPINAL INFECT10N WITH:TYZZER'S ORGANISヽ
1972]
4S
265
Rnsur-rs
1.
Clinical signs after intraspinal
inoculation.
Thirty five mice were divided into 5 groups of 7 mice, and each group was injected between the 3rd and the 4th lumbal vertebrae (1.v.) with infective homogenate containing 4xlOs, 4xl0+,4x10s, 4xl0z or 4X10 organisms. Observation was made for 20 days after inoculation. As shown in Tarr.B l, all the mice infected with 4x10s showed bristled-hairs on the next day, and 3 of 7 mice u,ere paralytic in a hind paw while one in both hind paws. Two days after inoculation 4 mice were cramped in the whole body, and 2 of them were also paralytic in one of fore-paws and both hind paws. Paralysis in either both hind paws or the trunk plus extremities was observed in 2 mice without any sign of cramp. Five animals Taer-s I Clinical signs ol mice after intraspinal inoculation between the 3rd and the 4th lumbal vertebrae. Number of
Mouse
organisms
No.
BPh* CPfbh
４ ５ ６ ７ １ ３ ４
BPbh
５ ６
BPh B
７
S
S
BPbh BPc BPbf
２
S
一
２ ４ ５ ６ ７
B: S: H: C: D:
一 一一一一
３
Clinical sign of each individual mouse.
ｈ 一 一 一 一 一 Ｐ 一
１
*
CPbf
一
一 一 一Ｓ 一
，
７
Ｂ 一
６
CPc
Ｓ
５
BPbh
D P D H P D
一Ｐ
Ｑリ
４
一一一一一
３
4× 102
CPc BPc CPbf Ａ ｂ
Ｓ︶
１
4× 103
BPs
P P P
Ｂ Ｂ
S
P P
Ｄ Ｈ Ｄ Ｄ Ｂ Ｂ 一Ｄ 一
２
B CPfbh B CPbh BPh P BPh P B CPc BPbh P B B
Ｄ Ｄ Ｄ Ｄ Ｄ
３
4× 104
Days after inoculation
12345678910
Ｐ Ｐ Ｐ Ｄ Ｐ Ｐ Ｄ
２
4× 105
１
inoculated
S
Pbh
Pbh
Pbh
Pbh
Pbh
Pbh
Pbh
Pbh
P: Whole body paralysis Ph: Paralysis in a hind-paw Hypersensitiveness Pbh: Paralysis in both hind-paws Pfbh: Paralysis in a fore-paw and both hind-paws Hunched-back Ps: Paralysis in the parts behind the shoulder Cramp Pbf: Paralysis in both fore-paws Dead Pc: Paralysis in the parts behind the neck
No signs Bristled hairs
266
T. ONODERA & K.
FUJIWARA
lYor.42.
surviving 3 days after inoculation were paralytic in the whole body and died on the next duy. Three days after inoculation with 4X104, two of 7 mice became paralytic in the whole body. Five days after inoculation, among 4 surviving animals, one showed cramp and paralysis in both fore-paws, while the remaining 2 the whole body paralysis. Only one animal survived for 20 days being hypersensitive arround one week after inoculation. With 4xl0s organisms, one of 7 mice was paralytic in both hind paws 3 days after inoculation and in the whole body 5 days after inoculation, dying on the following day. The remaining 6 mice survived through the experiment, though only one of them showed paralysis in both hind paws. With 4 x l0z organisms, only one of 7 mice showing paralysis in both hind paws on the 7th day died 19 days after inoculation. No sign of illness appeared in all mice after inoculation with 4 X l0 organisms. As described above, flaccid paralysis ascending with the development of spinal cord lesions appeared, and the incidence and the development of paralysis was found to be localizecl definitell, at a part of the body after inoculation with a smaller dose. Then, the following experiments were performed to see the relationship between clinical signs and the sites of intraspinal inoculation. Twenty-one mice were divided into 3 groups of 7 mice and each group was inoculated with 6 X 106 organisms at a level between the 3rd and the 4th 1.v., between the 5th and the 6th l.v. or between the llth and the 12th thoracic vertebrae (t.v.). Other 3 groups of 7 mice were infected with 6 x 10* organisms at the levels described above. As shown in TeerB 2 and 3, after inoculation with 6 x 106 organisms between the 5th and the 6th 1.v., all the mice showed bristled-hairs on the next day of inoculation. Two days after inoculation, one of them was paralytic in both hind-paws while another cramped in the whole body. Three days after inoculation, one of 6 surviving mice was hypersensitive, while four and one showed bristled-hairs, and the whole body paralysis, respectively. On the 4th day, 2 of 4 surviving mice showed bristled-hairs, and other 2 were hunchbacked. Five days after inoculation, one of 3 surviving mice showed hunched-back, and the remaining showed either cramp or paralysis resulting in collapse. A11 died within 6 days after inoculation. Three days afterinoculation with 6x106 organisms between the 1lth and the 12th t.v., one of 5 surviving mice were paralytic in the trunk and the extremities, and the remaining 4 were paralytic in the whole body. A11 the mice died within 5 days after infection. In mice inoculated between the 5th and the 6th 1.v., central neryous disorders were more prominent than paralysis in fore- or hind-paws. Mice having been inoculated between the llth and the l2th t.v. showed paralysis in the trunk and extremities without apparent ascending flaccid paralysis. Three days after inoculation with 6xlOa organisms between the 5th and the 6th 1.v., 3 of 7 mice showed paralysis in a hind-paw. Five days after inoculation, 3 mice shorved bristled hairs, while one and two paralysis in both hind-paws, and in the trunk plus extremities, respectively. On the seventh day, 2 of 4 surviving mice were hypersensitive, while one paralytic in both hind-paws and one hunch-backed. Two mice survived throughout the experiment showing hypersensitiveness. After 6 x 104 organisms were introduced between the 5th and the 6th 1.v., 4 of 7 mice died within l0 days showing apparent ascending flaccid paralysis. In contrast, such paralysis was not so apparent after inoculation with 6xlOa organisms between the llth and the 12th t.v., although all 7 mice died within 7 days showing acute whole body
ｒ ｒｒＦ⋮ ⋮ ⋮ ９４ ７′ Ｑ︶
INTRASPINAL INFECTION WITH TYZZER'S ORGANISMS
267
Tesr,E, 2
Clinical signs of mice after intraspinal inoculation with Mouse
Inoculation
２ ５ ６ ７
Ｄ Ｄ
Ｄ Ｄ Ｐ Ｄ Ｄ Ｐ
CPb-h
Ｐ
６
CPc
Ｐ
５ ７
B
２ ４
Ｄ Ｄ
６
一 一 一
５ ７
Ｄ Ｄ Ｐ Ｐ Ｄ
３
Ｄ Ｐ Ｐ Ｐ Ｒ Ｐ
ｓＣ ＤＰ Ｐ ＢＣ Ｃ
D
Ｂ Ｂ Ｂ Ｂ
１
See the foot-note on
C
Ｐ
４
*
CPc
Ｐ
３
and the I2th l.v.
CPs
Ｐ
２
Between the I lth
Ｐ
Ｂ Ｂ Ｂ Ｂ Ｂ Ｂ Ｂ
１
the 3rd and the 4th l.v.
6
Ｄ Ｄ Ｄ Ｄ
４
Between
5 Ｐ ＣＨ Ｂ
３
and the 6th l.v.
4
3
ｎ ︲ ︲ ｉ Ｃ， ｂ Ｂ Ｂ Ｂ Ｂ Ｄ Ｐ
Ｂ Ｂ Ｂ Ｂ Ｂ Ｂ Ｂ
１
Between the Sth
106 organisms.
ＤＤ Ｈ ＨＢ Ｂ
1
x
Days after inoculation
,
Ｈ Ｐ Ｂ ＢＢＢ
No.
6
Tealr l.
Tasr.E, 3
Clinical signs of mice after intraspinal inoculation with Inoculation
Mouse ２
ｈ ｂ Ｐ
２ ４
Ｂ Ｂ
５
Ｂ
６
Ｂ Ｂ
３ ７
B B
Ｂ
５
Ｂ
６
Ｄ Ｐ Ｄ
Ｂ
４
Ｂ
７
ｈ ｂＣＬＲＰＢＤ Ｐ
Ｂ
３
Ｂ
２
l.
ｈ Ｐ
ｈ 一 一 一 一 一 Ｐ 一
１
See the foot-note on Tanr-e
B
6
7
8
D
D
PD
9
10 Ｄ Ｓ Ｓ
Pbh
5
Ｐ Ｓ Ｄ Ｓ
1 Ｂ
１
*
Pbh Pbh
ｈ ｂＲＣ ＢＰＲ町Ｐ
７
the
llth
Ps
ｈ Ｒ Ｓ ｂ Ｓ Ｐ
６
Between
and the 12th l.v.
4
Ｄ Ｈ ｓ
５
the 3rd and the 4th l.v.
organisms.
Ｔ
３ ４
Between
3 B B Ph
ｈ Ｄ Ｄ ｂ Ｓ Ｐ Ｓ Ｓ Ｐ Ｄ Ｄ Ｄ Ｐ Ｐ
１
the 5th and the 6th l.v.
2
ｈ ｈ Ｒ Ｒ ｂ Ｂ ｂ Ｂ Ｂ Ｃ ＰＤ Ｐ Ｐ ＰＤ Ｄ Ｐ Ｐ Ｄ Ｄ Ｐ Ｐ Ｐ
1
一一一一一一一
こ o。
x l0{
Days after inoculation Ｆ 一 一 一 一 一 一 Ｂ 一
1ヽ
Between
6
268
T. ONODERA & K.
FUJIWARA
lYor-.42
paralysis.
In 9 mice having received 0.04 ml of PBS at the same sites as described above, two showed a persisting paralysis of both hind-paws. In case of injection with 0.02 ml PBS, however, all 9 mice showed a flaccidness in the posterior half of the body only for a short time after inoculation, and they recovered completely one hour later.
2.
Multiplicatton of organisms in the brain and spinal cord after intraspinal inoculation. Thirty five mice were inoculated intraspinally with 9 X 106 organisms at a site between the 3rd and the 4th 1.v.. Seven animals were killed each day following inoculation, and the number of organisms was estimated by a method described previously 14, l7). After infection with 9 X 106 organisms, the mean logarithmic numbers of organisms either per spinal cord or per brain reached a maximum 4 davs after infection (Tanrn 3). On smear preparations of affected tissues stained by thionin or Giemsa, organisms showed a pleomorphism. Some of them were about 2-5 pm (Type A), and 6-15 pm (Type B) long, both showing definite azurophile nuclear bodies. There were also thick organisms 10-15 pm long, densely stained with thionin or Giemsa stain without visible nuclear bodies (Type C). Spores or sporulating organisms (Type S) were also present. On the next day of inoculation, the A, B, and C types appeared almost in equal number in the brain or the spinal cord tissues. Two days after inoculatign, the organisms of type B were predominant in the spinal cord, while both B and C types were predominant in the brain. Three days after inoculation, all these bacillary types of organisms markedly decreased in number while there was a moderate increase in number of the S type. Similar observation was made after inoculation with 6 x 102 organisms using 49 mice. As shown in Tesr,E 4, the mean logarithmic number', of organisms per spinal cord showed a maximum 7 days after infection. In this case, the A and B types existed in equal number in the spinal cord 5 days after infection, while the B type was still numerous in the brain or the spinal cord 7 days after inoculation. From these results, the increase in the total number of organisms after inoculation with larger dose, seemed to reflect that of the B type. Spore-bearing organisms were always of the C type, while some non-spore bearing C type organisms were observed also in an early stage of infection. After inoculation with smaller dose, the C type organisms were very few.
3.
Light microscop2. Twenty mice were inoculated intraspinally with I x l0o organisms at a site between the 3rd and the 4th 1.v., and 2 each were killed 10, 30, +8, 76 and 96 hours after infection. Surviving animals were killed also 126, 150 and 170 hours alter inoculation. Forty-eight hours after inoculation, in one of examined animals showing paralysis in hind-paws, shrinkage and degeneration of neurons were observed in the lumbar spinal cord rvith infiltration of neutrophiles and mononuclear round cells near the foci (Photo 1). Rod-shaped organisms stained within nerve cells as well as extracellularly. The other one killed at the same time showed hemiplegia, and necrotic lesions were observed in the cervical or thoracic cord with some cellular infiltrations. Seventy-two hours after inoculation, histopathologic findings were not so much different from those observed 48 hours after inoculation, while the animals showed ataxia and paralysis in the parts behind the neck. There were atrophy of neurons, karyorrhexis of astrocytes and capillary endothelial cells (Photo 2), karyopyknosis of oligodendroglia cells, degeneration and erosion of the central canal ependyma, demyelination in the white
INTRASPINAL INFECTION WITH:TYZZER'S ORGANIS]MiS
1972]
l[｀
ABLE 4
Nurnber of organisnls in the brain and thc spinal cord a■
2
organisms
70*
5.00 く(5.00 5.48 5.70 く〔5.00 5.60
5.48 6.08 6.04 5.90 5.60 5.90 6.08
く〔5.35**
5.87
5,90
6.18 6.08 6。 18 5.83
5
6.36
6.70 6.67 6.94 6.79 6.87 6.65
6.41
6.71
6.36 6.59 6.32
6.38 6.32
5.48 5。
74
5.78
く (5.00
6.48 6.18 6.26 6.04 6.20 5.70
60
5.85 5,90
6.07
6.39
6。 76
5。 98
6.23
5。 78
く (5.00
5.00
く〔5.00 く〔5.00 く〔5.00
く〔5.00 く(5.00
