- Email: [email protected]
Porcine congenital tremor type AII: Spinal cord morphometry
Br. vet . .7. (1986) . 142, 1 4 5
PORCINE CONGENITAL TREMOR TYPE AM SPINAL CORD MORPHOMETRY
J . T . DONE, J. WOOLLEY, D . H . UPCOTT and C . N . HEBERT Central Veterinary Laboratory, Weybridge, Surrey, England KT153NB
SUMMARY Semi-automated planimetry was used to determine cross-sectional areas of spinal grey and white matter of baby piglets by direct microscopy of Woelckestained sections at levels C3 , T 1 _2, T7 _ 8 and L5_6 . The specimens came from 16 cases of porcine congenital tremor type All (CT AII) experimentally induced by infecting sows, when 30 days pregnant, with the Weybridge CT AII agent (presumed to be a virus) and from nine normal controls of the same age . Affected piglets showed mean reductions in spinal cord cross-sectional areas of about 5% for grey matter, 15% for white matter and 12% for grey and white matter combined . The reduction was apparent at all four levels of the spinal cord studied. However this pattern did not differ in a diagnostically useful way from that seen in congenital tremor type AI (CT AI), after allowing for the potentially confounding effects of within-litter variation and varying gestation age at time of exposure . INTRODUCTION The porcine congenital tremor (CT) syndrome comprises several aetiologically distinct entities with overlapping pathological patterns but with a characteristic symptom (i.e . tremor) in common . For convenience, entities within the syndrome have been classified (Done, 1976a and b, 1982 ; Done & Bradley, 1981) as type A (a form of CT with defined pathological characters and known aetiology) or type B (a form of CT as yet inadequately characterized and/or of unknown aetiology) . Type A includes two forms caused by transplacental virus infection during gestation ; CT AI caused by certain strains of swine fever virus (Pestivirus suis), and CT All caused by an unrelated but unknown virus . Both forms can be reproduced experimentally . Conventionally the forms of CT have been defined pathologically in terms of morbid anatomy, histology and ultrastructure ; and the late Deryck Patterson and his colleagues contributed an invaluable additional dimension to the diagnostic taxonomy of CT through their work on the neurochemistry of congenital nervous disorders of farm animals (see Patterson, 1977 ; Patterson & Done, 1977 ; Sweasey & Patterson, 1980) . In their original study of experimentally-induced CT AI, Done & Harding (1969) noted a quantitative reduction in the cross-sectional area of the spinal cord in affected piglets ; and a similar but smaller reduction has been observed in CT AII (J . T. Done, A .
1 46
BRITISH VETERINARY JOURNAL, 142, 2
Rogers & D . E . Wells, unpublished) . More recently, the availability of improved techniques has prompted a re-examination of the CT AI material in more detail (Done et al., 1984). The present paper reports a comparable study on available material from experimentally-induced cases of CT AII for comparison with CT AI and to extend the data base available for diagnostic taxonomy of the CT syndrome . MATERIALS AND METHODS
The pathological material used was derived from experimental transmission studies carried out in 1966-7 (J . T . Done & J . D . J . Harding, unpublished), supplemented by control material common to the studies of CT AI and CT AII . It comprised standard sets of tissues embedded in paraffin wax . Full protocols of the experiments were available . The technical methods were essentially the same as used by Done et al. (1984) in their study of CT Al .
Source of animals The origin of the piglets is summarized in Table I . All were sired by the same Large White (LW) boar out of closely-related LW sows . Sow 3504 provided all the normal control piglets as well as one of the affected litters . The two trembling litters resulted from intramuscular infection of their pregnant dams with bacterially-sterile emulsions of/CNS tissue from affected piglets of passages 1 and 2 ; i .e . the material represents passages 2 and 3 of the causal agent of CT All . All the piglets in both the affected litters showed congenital tremor of varying severity . Table I Origin of the piglets used in study of congenital tremor type AII Litter
No. of piglets
A (3504/2) B (3463/2) Cl (3504/1) C2 (3504/3)
11* 5*
31 30
i .m . i .m .
2 3
4+ 5+
-
-
Normal controls Normal controls
Maternal exposure Gestation day Route
Passage
* All these pigs were clinically affected with congenital tremor of varying severity . +These are the same normal controls as used in the study on CT AI by Done et al (1984) . Groups of four to 11 piglets from each litter were killed within 24 h of birth by exsanguination under deep sodium pentobarbitone anaesthesia, and fixed in 10% formol calcium by whole-body perfusion followed by immersion for at least one week . Litter mates of each group were used for preliminary neurochemical studies (see Patterson et al., 1976) .
Pathological material
After fixation, the piglets were dissected, weighed and measured, and standard blocks of the CNS were embedded in paraffin wax. As part of the standard CNS series transverse
PORCINE CONGENITAL TREMOR TYPE All 147 blocks of spinal cord, with dorsal root ganglia attached, were taken at segments C3, TI-21 T-_s and L5, . For the purpose of this study, 20 pm paraffin sections from each block of spinal cord were stained by Woelcke's method for myelin . Microscopy Stained sections were examined by transmitted light in a binocular microscope (Reichert Neopan) with a camera lucida attachment, using a X1 . 6 or 2 . 5 objective and X 10 periplanetic oculars . Planimetry The outlines of the grey and white matter were traced by the cursor of a digitizing tablet which was linked to a microcomputer (Tektronix 4051) driven by an appropriate morphometric program (General Digitizing System 1 : Graphic Information Systems Ltd, Blairgowrie, Scotland) . The trace was displayed on a VDU, and the derived area measurements, after manipulation, were dumped to a printer . Routinely a single histological section at each spinal cord level from each pig was measured at least three times by a single operator .
RESULTS The estimated cross-sectional areas for grey matter, white matter and whole cord at each of the four cord levels are presented as litter means in Table II . The data have been aggregated for normal controls, since the means for the two litters did not differ significantly . The mean areas of grey matter of the affected litter, B, closely resembled those of the controls at all levels of the cord, but in litter A a significant reduction in size occurred at levels T 1 _ 2 (P< 0 . 05), and L 5_6 (P< 0 . 01), and in the mean of all levels (P< 0 . 05) . There was a very highly significant reduction in mean area of white matter at all levels in both affected litters compared with the controls (P< 0 . 001) and over the spinal cord as a whole the mean reduction in area was highly significant in litters A (P<0 . 001) and B (P<0-01). 1000-
• •
950
+
900
+
++
X
850
+
800
• z
750
+ *
+41$, +
* -*
0 0 ** 00 • 700 * • 650 3 600 1 I I 1 1 1 300 350 400 450 500 550 600 Grey matter (mm 2 x 100) Fig. 1 . Individual means (all levels combined) for spinal cord cross-sectional areas of white matter in relation to grey matter in affected and normal control piglets . + Normal controls, * affected litter A, o affected litter B.
BRITISH VETERINARY JOURNAL, 142, 2
-i .-
b2
ó
r
00 u~ M
H
c, M oo cl] CD <.dr- r- 00
00
^ cl e,
~1 N M M r
íU ')
1* 1*
~t
O
w
al en
M
7
M
Mn
h G1
b)
tr
W F7 -,l- r °o r o oo ó ~o
R
n á o0 oó
°O
z V
bA M. V C C
a 0 Us
C rt
N Cd
O)
n
M
tF N C` C' íNn'C'o
N M
7
`.
r-
8 X
n r-- cl 01
E E z Vvw
V
(~1
Jo r
V
v
m
v t -n'Q "
Û
~v
á
~
•cd• E
• rti Ln C\
v c.ó0 ,n N
y v d
N N M
O
b C
M ~
Q W U
c C O ~~ on O w T mz r. UM U 7J C C pp
r- u ° C cO û y 4 • O,~b a„ cvC• tC0 C ,~ ~5 >F- -"J
N y :0 C ~v-`C+°
PORCINE CONGENITAL TREMOR TYPE All
1 49
The relationship of grey and white matter areas for all levels of the cord combined, in individual piglets, is shown in Fig . 1 . Mean white matter areas for each litter, after adjustment by covariance analysis to allow for differences in grey matter, are given in Fig. 2, together with those found in litters of similar gestational age, but affected with CT AI . These (CT AI) litters have been previously reported by Done et al. (1984) and are included here for comparison with CT AII .
O O X E
E
d
900'-
CT All
Controls
860 820
CT Al
T
780 -
T
740-
1
E
q) 700t ran
1 T i
1
620
Tn
v a
580 540 500
A
B
C
D,E,F
Mean white matter spinal cord cross-sectional areas and 95% confidence limits, after adjustment for variations in grey matter area . Comparison of CT AII affected litters, CT AI litters of similar gestational age, and normal controls . Fig. 2 .
DISCUSSION
As Table II indicates, in trembling piglets there was an overall reduction in spinal cord cross-sectional areas of about 12% . There was little change in grey matter area, particularly in litter B piglets, but there was an overall reduction of some 15% in the area of white matter. While there was some variation within litters (see Fig . 1), the overall pattern was similar at all four levels of the cord . Compared with a fall in extractable lipids of about 37% in the spinal cord of pigs similarly affected with CTA II (Patterson et al, 1976), the reduction in white matter area was relatively small . However, it is necessary to remember that the spinal white matter does not consist solely of myelin (see Foulkes & Patterson, 1974), and it is interesting to note that a similar discrepancy was found in CT AI (Done et al., 1984) . Much of the discussion in that paper on spinal cord size and composition in CT AI is relevant to CT All, and does not need to be repeated here . The overall reduction in spinal cord cross-sectional areas in CT AII (12%) was less than that found in piglets exposed to the CT AI agent (21%), but the difference was not statistically significant. Although the absolute reduction in white matter area was greater in CT AI, there was little difference in the relationship of white to grey matter. In terms of spinal cord morphometry, the two conditions do not differ from each other in pattern and extent in such a way as to be diagnostically useful when the potentially confounding effects of
1 50
BRITISH VETERINARY JOURNAL, 142, 2
within-litter variation and varying gestational age at time of exposure are taken into account (Fig . 2) . As remarked elsewhere (Done et al., 1984), probably the best use of such data is as a component of a multi-dimensional image of the condition under investigation . And, as studies on swine fever (CT AI) and other viral teratogens have shown, the image may be expected to vary with the weight and the timing of infection as well as with the identity of the teratogen and the genetic liability of the host .
ACKNOWLEDGEMENTS The authors are indebted to the late Mr J. D . J . Harding who played a significant part in the early investigations of this condition and in the provision of the material used ; to Mrs E . Cowlard, Mrs A . Rogers and Miss D . Wells for their care of the animals and the archives ; to Mrs L . Heath and Mr J . Bailey for technical assistance ; to Mr C . Allen for data handling and production of the figures and to Mrs J . Cox for her help in preparing the manuscript .
REFERENCES T . (1976a) . In The Veterinary Annual, 16th issue, ed . C . S . G . Grunsell and F . W . G . Hill, p . 98 . Bristol : Wright Scientechnica . DONE, J . T . (1976b) . Advances in Veterinary Science and Comparative Medicine 20, 69 . DONE, J . T . (1982) . In Diagnostico de las enfermedades del cerdo, ed . R. Ramirez Necoechea and C . Pijoan Aguade. Mexico : Ramirez and Pijoan . DONE, J . T . & BRADLEY, R. (1981) . In Dunne's Diseases of Swine, 5th edn, ed . A . D . Leman, R. D Glock, W . L. Mengeling, R . H . C . Penny, E . Scholl and B . Straw, p . 52 . Ames . Iowa : State University Press . DONE, J . T . & HARDING, J . D . J . (1969) . Excerpta Medica International Congress Series 173, 112 . DONE, J . T ., WOOLLEY, J ., UPco9'"l', D . H . & HEBERT, C . N . (1984) . Zentralblatt far Veterinärmedizin, Reihe A 31, 81 . FOULKES, J . A . & PA 1- I'ERSON, D . S . P . (1974) . Brain Research 82, 139 . PATTERSON, D . S . P . (1977) . In Infections and Pregnancy, ed . C . R . Coid, p . 307 . London, New York and San Francisco : Academic Press . PAI TERSON, D . S . P . & DONE, J . T. (1977) . British Veterinary Journal 133, 111 . PA I'I'ERSON, D . S . P ., DONE, J . T., FOU LKES, J . A . & SWEASEY, D . (1976) . Journal of Neurochemistry 26, 481 . SWEASEY, D . & PATTERSON, D . S . P. (1980). In Animal Models of Neurological Disease, ed . F . C . Rose and P . O . Benham, p . 306 . Tunbridge Wells : Pitman Medical Ltd . DONE, J .
(Accepted for publication 1 February 1985)
PORCINE CONGENITAL TREMOR TYPE AM SPINAL CORD MORPHOMETRY
J . T . DONE, J. WOOLLEY, D . H . UPCOTT and C . N . HEBERT Central Veterinary Laboratory, Weybridge, Surrey, England KT153NB
SUMMARY Semi-automated planimetry was used to determine cross-sectional areas of spinal grey and white matter of baby piglets by direct microscopy of Woelckestained sections at levels C3 , T 1 _2, T7 _ 8 and L5_6 . The specimens came from 16 cases of porcine congenital tremor type All (CT AII) experimentally induced by infecting sows, when 30 days pregnant, with the Weybridge CT AII agent (presumed to be a virus) and from nine normal controls of the same age . Affected piglets showed mean reductions in spinal cord cross-sectional areas of about 5% for grey matter, 15% for white matter and 12% for grey and white matter combined . The reduction was apparent at all four levels of the spinal cord studied. However this pattern did not differ in a diagnostically useful way from that seen in congenital tremor type AI (CT AI), after allowing for the potentially confounding effects of within-litter variation and varying gestation age at time of exposure . INTRODUCTION The porcine congenital tremor (CT) syndrome comprises several aetiologically distinct entities with overlapping pathological patterns but with a characteristic symptom (i.e . tremor) in common . For convenience, entities within the syndrome have been classified (Done, 1976a and b, 1982 ; Done & Bradley, 1981) as type A (a form of CT with defined pathological characters and known aetiology) or type B (a form of CT as yet inadequately characterized and/or of unknown aetiology) . Type A includes two forms caused by transplacental virus infection during gestation ; CT AI caused by certain strains of swine fever virus (Pestivirus suis), and CT All caused by an unrelated but unknown virus . Both forms can be reproduced experimentally . Conventionally the forms of CT have been defined pathologically in terms of morbid anatomy, histology and ultrastructure ; and the late Deryck Patterson and his colleagues contributed an invaluable additional dimension to the diagnostic taxonomy of CT through their work on the neurochemistry of congenital nervous disorders of farm animals (see Patterson, 1977 ; Patterson & Done, 1977 ; Sweasey & Patterson, 1980) . In their original study of experimentally-induced CT AI, Done & Harding (1969) noted a quantitative reduction in the cross-sectional area of the spinal cord in affected piglets ; and a similar but smaller reduction has been observed in CT AII (J . T. Done, A .
1 46
BRITISH VETERINARY JOURNAL, 142, 2
Rogers & D . E . Wells, unpublished) . More recently, the availability of improved techniques has prompted a re-examination of the CT AI material in more detail (Done et al., 1984). The present paper reports a comparable study on available material from experimentally-induced cases of CT AII for comparison with CT AI and to extend the data base available for diagnostic taxonomy of the CT syndrome . MATERIALS AND METHODS
The pathological material used was derived from experimental transmission studies carried out in 1966-7 (J . T . Done & J . D . J . Harding, unpublished), supplemented by control material common to the studies of CT AI and CT AII . It comprised standard sets of tissues embedded in paraffin wax . Full protocols of the experiments were available . The technical methods were essentially the same as used by Done et al. (1984) in their study of CT Al .
Source of animals The origin of the piglets is summarized in Table I . All were sired by the same Large White (LW) boar out of closely-related LW sows . Sow 3504 provided all the normal control piglets as well as one of the affected litters . The two trembling litters resulted from intramuscular infection of their pregnant dams with bacterially-sterile emulsions of/CNS tissue from affected piglets of passages 1 and 2 ; i .e . the material represents passages 2 and 3 of the causal agent of CT All . All the piglets in both the affected litters showed congenital tremor of varying severity . Table I Origin of the piglets used in study of congenital tremor type AII Litter
No. of piglets
A (3504/2) B (3463/2) Cl (3504/1) C2 (3504/3)
11* 5*
31 30
i .m . i .m .
2 3
4+ 5+
-
-
Normal controls Normal controls
Maternal exposure Gestation day Route
Passage
* All these pigs were clinically affected with congenital tremor of varying severity . +These are the same normal controls as used in the study on CT AI by Done et al (1984) . Groups of four to 11 piglets from each litter were killed within 24 h of birth by exsanguination under deep sodium pentobarbitone anaesthesia, and fixed in 10% formol calcium by whole-body perfusion followed by immersion for at least one week . Litter mates of each group were used for preliminary neurochemical studies (see Patterson et al., 1976) .
Pathological material
After fixation, the piglets were dissected, weighed and measured, and standard blocks of the CNS were embedded in paraffin wax. As part of the standard CNS series transverse
PORCINE CONGENITAL TREMOR TYPE All 147 blocks of spinal cord, with dorsal root ganglia attached, were taken at segments C3, TI-21 T-_s and L5, . For the purpose of this study, 20 pm paraffin sections from each block of spinal cord were stained by Woelcke's method for myelin . Microscopy Stained sections were examined by transmitted light in a binocular microscope (Reichert Neopan) with a camera lucida attachment, using a X1 . 6 or 2 . 5 objective and X 10 periplanetic oculars . Planimetry The outlines of the grey and white matter were traced by the cursor of a digitizing tablet which was linked to a microcomputer (Tektronix 4051) driven by an appropriate morphometric program (General Digitizing System 1 : Graphic Information Systems Ltd, Blairgowrie, Scotland) . The trace was displayed on a VDU, and the derived area measurements, after manipulation, were dumped to a printer . Routinely a single histological section at each spinal cord level from each pig was measured at least three times by a single operator .
RESULTS The estimated cross-sectional areas for grey matter, white matter and whole cord at each of the four cord levels are presented as litter means in Table II . The data have been aggregated for normal controls, since the means for the two litters did not differ significantly . The mean areas of grey matter of the affected litter, B, closely resembled those of the controls at all levels of the cord, but in litter A a significant reduction in size occurred at levels T 1 _ 2 (P< 0 . 05), and L 5_6 (P< 0 . 01), and in the mean of all levels (P< 0 . 05) . There was a very highly significant reduction in mean area of white matter at all levels in both affected litters compared with the controls (P< 0 . 001) and over the spinal cord as a whole the mean reduction in area was highly significant in litters A (P<0 . 001) and B (P<0-01). 1000-
• •
950
+
900
+
++
X
850
+
800
• z
750
+ *
+41$, +
* -*
0 0 ** 00 • 700 * • 650 3 600 1 I I 1 1 1 300 350 400 450 500 550 600 Grey matter (mm 2 x 100) Fig. 1 . Individual means (all levels combined) for spinal cord cross-sectional areas of white matter in relation to grey matter in affected and normal control piglets . + Normal controls, * affected litter A, o affected litter B.
BRITISH VETERINARY JOURNAL, 142, 2
-i .-
b2
ó
r
00 u~ M
H
c, M oo cl] CD <.dr- r- 00
00
^ cl e,
~1 N M M r
íU ')
1* 1*
~t
O
w
al en
M
7
M
Mn
h G1
b)
tr
W F7 -,l- r °o r o oo ó ~o
R
n á o0 oó
°O
z V
bA M. V C C
a 0 Us
C rt
N Cd
O)
n
M
tF N C` C' íNn'C'o
N M
7
`.
r-
8 X
n r-- cl 01
E E z Vvw
V
(~1
Jo r
V
v
m
v t -n'Q "
Û
~v
á
~
•cd• E
• rti Ln C\
v c.ó0 ,n N
y v d
N N M
O
b C
M ~
Q W U
c C O ~~ on O w T mz r. UM U 7J C C pp
r- u ° C cO û y 4 • O,~b a„ cvC• tC0 C ,~ ~5 >F- -"J
N y :0 C ~v-`C+°
PORCINE CONGENITAL TREMOR TYPE All
1 49
The relationship of grey and white matter areas for all levels of the cord combined, in individual piglets, is shown in Fig . 1 . Mean white matter areas for each litter, after adjustment by covariance analysis to allow for differences in grey matter, are given in Fig. 2, together with those found in litters of similar gestational age, but affected with CT AI . These (CT AI) litters have been previously reported by Done et al. (1984) and are included here for comparison with CT AII .
O O X E
E
d
900'-
CT All
Controls
860 820
CT Al
T
780 -
T
740-
1
E
q) 700t ran
1 T i
1
620
Tn
v a
580 540 500
A
B
C
D,E,F
Mean white matter spinal cord cross-sectional areas and 95% confidence limits, after adjustment for variations in grey matter area . Comparison of CT AII affected litters, CT AI litters of similar gestational age, and normal controls . Fig. 2 .
DISCUSSION
As Table II indicates, in trembling piglets there was an overall reduction in spinal cord cross-sectional areas of about 12% . There was little change in grey matter area, particularly in litter B piglets, but there was an overall reduction of some 15% in the area of white matter. While there was some variation within litters (see Fig . 1), the overall pattern was similar at all four levels of the cord . Compared with a fall in extractable lipids of about 37% in the spinal cord of pigs similarly affected with CTA II (Patterson et al, 1976), the reduction in white matter area was relatively small . However, it is necessary to remember that the spinal white matter does not consist solely of myelin (see Foulkes & Patterson, 1974), and it is interesting to note that a similar discrepancy was found in CT AI (Done et al., 1984) . Much of the discussion in that paper on spinal cord size and composition in CT AI is relevant to CT All, and does not need to be repeated here . The overall reduction in spinal cord cross-sectional areas in CT AII (12%) was less than that found in piglets exposed to the CT AI agent (21%), but the difference was not statistically significant. Although the absolute reduction in white matter area was greater in CT AI, there was little difference in the relationship of white to grey matter. In terms of spinal cord morphometry, the two conditions do not differ from each other in pattern and extent in such a way as to be diagnostically useful when the potentially confounding effects of
1 50
BRITISH VETERINARY JOURNAL, 142, 2
within-litter variation and varying gestational age at time of exposure are taken into account (Fig . 2) . As remarked elsewhere (Done et al., 1984), probably the best use of such data is as a component of a multi-dimensional image of the condition under investigation . And, as studies on swine fever (CT AI) and other viral teratogens have shown, the image may be expected to vary with the weight and the timing of infection as well as with the identity of the teratogen and the genetic liability of the host .
ACKNOWLEDGEMENTS The authors are indebted to the late Mr J. D . J . Harding who played a significant part in the early investigations of this condition and in the provision of the material used ; to Mrs E . Cowlard, Mrs A . Rogers and Miss D . Wells for their care of the animals and the archives ; to Mrs L . Heath and Mr J . Bailey for technical assistance ; to Mr C . Allen for data handling and production of the figures and to Mrs J . Cox for her help in preparing the manuscript .
REFERENCES T . (1976a) . In The Veterinary Annual, 16th issue, ed . C . S . G . Grunsell and F . W . G . Hill, p . 98 . Bristol : Wright Scientechnica . DONE, J . T . (1976b) . Advances in Veterinary Science and Comparative Medicine 20, 69 . DONE, J . T . (1982) . In Diagnostico de las enfermedades del cerdo, ed . R. Ramirez Necoechea and C . Pijoan Aguade. Mexico : Ramirez and Pijoan . DONE, J . T . & BRADLEY, R. (1981) . In Dunne's Diseases of Swine, 5th edn, ed . A . D . Leman, R. D Glock, W . L. Mengeling, R . H . C . Penny, E . Scholl and B . Straw, p . 52 . Ames . Iowa : State University Press . DONE, J . T . & HARDING, J . D . J . (1969) . Excerpta Medica International Congress Series 173, 112 . DONE, J . T ., WOOLLEY, J ., UPco9'"l', D . H . & HEBERT, C . N . (1984) . Zentralblatt far Veterinärmedizin, Reihe A 31, 81 . FOULKES, J . A . & PA 1- I'ERSON, D . S . P . (1974) . Brain Research 82, 139 . PATTERSON, D . S . P . (1977) . In Infections and Pregnancy, ed . C . R . Coid, p . 307 . London, New York and San Francisco : Academic Press . PAI TERSON, D . S . P . & DONE, J . T. (1977) . British Veterinary Journal 133, 111 . PA I'I'ERSON, D . S . P ., DONE, J . T., FOU LKES, J . A . & SWEASEY, D . (1976) . Journal of Neurochemistry 26, 481 . SWEASEY, D . & PATTERSON, D . S . P. (1980). In Animal Models of Neurological Disease, ed . F . C . Rose and P . O . Benham, p . 306 . Tunbridge Wells : Pitman Medical Ltd . DONE, J .
(Accepted for publication 1 February 1985)