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pregnancy: ...
AN INVESTIGATION OF MOSAIC / CHIMAERISM IN THE DOMESTIC HORSE ( Eguu s cabal lus caba l lu s )
A thesi s pre sented i n part i a l
f u l f i lment of the requi rements for the degree of Doc tor of Phi losophy in
Cytogeneti c s at Ma s s ey Unive r s i ty
Karen Shei la Wa lker 1980
. t• I• f
• ' J- ••
ABSTRACT
Thi s s tudy invo lved three as pect s of the f i e l d of Cytogenet i c s app lied to the dome s t i c horse (�. caba l lus caba l lu s ) . The f i r s t as pect , the iden t i f ic at ion and investigat ion o f individua l s with cytogen e t i c anoma l i e s , i s fundamental to Cytogenet ic s . The individ
ual s di scovered , however , pointed to t he c lose interre l at i onshi p of Cyt ogenet i c and Genet i c re search. Thirdly , que s t ions of fert i l i t y , and in one mare the t en tat ive demon strat ion of an app l ication of cytogen e t i c s in veteri nary c l inic a l eva luati on , were concerned wi th fun c t ions of C l in i c a l Cytogenet i c s .
The pr imary hypo thesi s of thi s study was that individual s w i th abnorma l sex chromosome con s t i tution s , the maj o r i ty of whi ch are e i ther mosa i c s or chimaeras , form a signi fi cant proport ion of some
�. £· caba l l us breed s , in part icu lar the Thoroughbred Breed . Sub s i d iary to that hypothes i s was that these mo sai c /chimaeras as a group were d i sproport i onat e ly responsib l e for the sub - fert i l i ty of some hor s e popu l at io ns , through infert i l i ty , sub - ferti l i ty and generating abnorma l off spring .
Five hors es were inve s tigated . One , a XY /XYY /?XO mo saic / chimaeric st a l l ion was ful ly ferti le . However , he s i r ed an XO s t i l l - born
fi l ly . Limi t ed stud i e s of three of his relatives and three of hi s o ther progeny , sugges ted the po ssib i l i ty of cytogenetic anoma l ie s in
tha t kinshi p . He was the f i rs t reported s ta l l i on with an XYY line , and his XO fi l ly was the f i r s t reported s ti l l -born foal with a chromo
some abnorma l i ty .
Two 64 , XX / 65 , XXY mares wi th no physical signs of intersexua l i ty were s tudied . One was infert i l e , however , there was insufficient info rmation to assess the o ther mare ' s fert i l i ty . An infert i l e mare was di scovered and inves tigated in depth , whi ch had 65 , XXX/65 , XXY / 64 , XX/ 6 6 , XXY / 6 6 , XXXX mixopl oid mosaic/ chimaeri sm , al though she was primari ly a tri p l e-X ma re . The fourth mare wa s primari ly thought to
be a pro l i f e rative mo sai c , whi c h exhi b i t ed mitotic ins tabi l i ty . P o s t mortem evi den c e , however , confi rmed that she had been cytogenetical ly abnorma l and that she probably was a mosai c / chimaera.
Aside f rom one XX/ XXY mare , whi ch had been born wi th congenital defec t s , the re was no thing ei ther in t he hi story or phenotype of these f ive hor s e s to dis tingui sh them f rom o ther Thoroughbreds. Only one
i i i
o f the mar e s was presented fo r ana l ys i s because of inferti l i ty , al though two of the o the r mares mi ght have been termed " shy breeders".
The d i s c overy of al l the ma j o r sex chromosome aneupl oid cond i t ions infrequent l y f ound in human s , XO , XXY , XYY and XXX , in the mo sai c / chi maeric cond i t i o n among l e s s than 200 Thoroughbreds eva luated , sugges ted
that chromo some anoma l ies are preva l ent in the Thoroughbred populat ion.
ACKNOWLEDGEMENTS
I am i ndebted to the Fac u l ty of Veter inary Science , Mas sey U niver s i ty , for providing the oppo rt un i ty and faci l i t i es for thi s i nvesti ga t i on .
In par t icular I wou l d l i ke to thank my supervisor Prof . A . N . B ruere and ac knowl edge the patient a s s i s tance and guidance of bo th \
the academi c and technical s taff of the Depar tment of Veterinary S c ienc e , wi th mention of : Prof . E . D . Fie lden , Dr . B . E . Gou l den , Dr . R . E . Harr i s , Dr . R . J . Ho l me s , Dr . I . L . Anderson , Dr . H . G . P earce , Mrs . R . B . Foo t e , Ms Karen Armi tage , and the staff of the Large Ani ma l Uni t .
Recogni t ion i s al s o due to the .Depar tment of Ve terinary Path
o logy and P ub l i c Hea l t h s taff and fac u l ty , wi th spec i a l mention of : M s L. Frey , the H i s to l ogy Preparation Uni t , the Po s t - mo rtem Examin
a t i on Fac i l i ty , Dr . A . C . Johns t one , Dr . M . R. Al ley , Dr . K . M . Moriarty , Dr . A . J . Robinson an d P . Ramadas s , Ph.D. candidate ; and P rof . •R . E . Munford and Mr . M . J . B i r t l e s of the Depar tment of Anatomy and Physiol ogy .
Thi s s tudy wou l d not have been pos s i b l e wi thout the he l p and c ooperation of horse owners and breeders in Ne\v Zeal and , to whom the au thor expresses grat itude . In add i t ion , the ac tive par t i c i pation of members of the veter inary profes sion was an invaluabl e hel p , espec i a l ly M r . H . Dewe s , M r . J . Hope , M s G . Verkerk , M s J . Franc i s , M r . P . Wi l l iams and Mr . C . Kel ly . I shou l d al s o l i ke to acknowledg e the f inanc i a l
a s s i s t anc e of The New Zea land Equine Re search Foundation .
I wou l d l i ke to expre s s my ap preci at ion to the Li brar ians and M r . T . Law (Photographi c Uni t ) f o r the i r t o l e rance and professiona l i sm , a s wel l as to the Department o f Bo tany and Zoo logy f o r thei r support and sufferance .
For their hel p , encouragement and endur ing fai th , a very spec i a l thankyou t o my paren t s .
Many peo pl e , known and unknown , have made thi s s tudy po s s ib l e . To them,
et.�<:>�.
C"CL ouJ<G o� �0(,. ·Mahalo .TABLE OF CONTENTS
Page Acknowl edgement s . . . iv Conten t s . . . . . . • . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . • • . . . . . . . v L i s t o f F i gures • . . . • . . . . . . . . . . . . . . . • . • . . . . . . . . . . . . . • . . • • . . . . . . • xi i Lis t o f Tab l es . . . . . . • • . . . . . . . . • . . . • • . . . . . . . . . . . . . . . xv
CHAPTER I - Introdu c tion 1
1.1. Inf er t i l i ty in the domes t i c hor se ( E . c abal lus cabal lus):
t h e aims of thi s study • . . . . . . . . . . . . . . . • • • . . . . . . . . . . . . . 3 1.2. Mosai c i sm and chimaer i sm : defini tions and pos s i b l e
r e l a t i o n to infert i l i ty . . . 5 1.2.1. Mosai c i sm , chimaeri sm and " mo sai c / chimaeri sm" :
def i n i tions . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . 5 1.2.2. Chimaeri sm: types , po s s i b l e relation to fert i l -
i ty and po s s i b l e pred i s po sing fac tors . .. . . . .. . . 6
1.2.2.1. Haemo po i e t i c chimaer i sm . . . .. . . . . 6 1. 2. 2. 2. Who le body chimaeri sm . . . . . . . . . . . . . . . . . . 7 1.2.3. Mo sai c i sm: pos s i b l e imp l i cat ions in infert i l i ty
and po s s i b l e pred i s po s i ng fac tors ..•.. . . . .... . . 7 1.2.3.1. Facto r s whi c h might pred i spo s e t o
format ion of mo saic i sm .... ... ...• . .·. . • . 8
1.2.3.2. The format i on of mo saic i sm . . . 9 1.3. Factor s affec t i ng mo sai c / chimaer i c compo s i tion and t hei r
interpretat i o n • . • • . . . . . . • • • • . . . . . . . . . • • • • • . . . . . . . . • • . . . . . 10
1 . 4 . Aspec t s o f the cytogenet i c evaluation o f mo sai c / chimaeri sm •.. 1 0 1 . 4 . 1 . Chromos ome preparati ons from cul tured c e l l s in
relat ion to the in- vivo mosai c / chimaeric condi tion 1 0 1 . 4 . 1 . 1 . Repres entat iveness o f the samp l e ...... ..... 1 1 1 . 4 . 1 . 2 . Di ffer ential viabi l i ty and growth of
c e l l l i nes in c u l ture ....• ................. 1 2 1 . 4 . 1 . 3 . Natura l , ar t i fac tua l and symptomati c
aneu p l o idy and hyperp loidy ........ ......... 1 2 1 . 4 . 1 . 4 . Mi togenic s ti mu l at ion and mi toic
i nhi b i t i on .. ................ ........... ... 1 3 1 . 4 . 2 . Mo sai c / chimaeri s m and sex- chromatin s tudies ...• ..•... 1 3
1 . 5 . Atypical deve lopment and cytogeneti c abnorma l i t i e s ..• .... .... 1 5 1 • 6 . The fami l y Egui dae . . . . . • . . . . . . . . . • . • • . • . . . . . . . . • . . . . . . . . . 1 6 1 . 6 . 1 . The karyotypes o f Equidae . . . . . . . • . . . . . . • . • • . • • . . . . . . • 1 6 1 . 6 . 2 . E quine hybri d s • . ...•• ......••• .•.....• .•• • .......• •.. 1 7 1 . 7 . Cytogenetics of�· caba l l u s caba l l u s ....•.... ....• • • • � • ....•. 1 9 1 . 7 . 1 . The norma l karyotyp e of�· £· cabal lus ••••........•• . 1 9 1 . 7 . 1 . 1 . Chromo s ome iden t i fi ca tion .•.• ........•.. ... 1 9 1 . 7 . 1 . 2 . Chrorao some po lymo rphi sms .....•• ........• ... 20 1 . 7 . 1 . 3 . The karyo type of the dome s t i c horse ... .... . 20 1 . 7 . 1 . 4 . Arrangement of the karyotype o f the
dome s t i c horse .. ..... ... .. . .... .. ..... . .• .. 2 1 1 . 7 . 2 . Abnormal karyo types in the dome s t i c hor se .... ... ... .. 2 1 1 . 7 . 3 . Infert i l i ty and chromosome anoma l i e s in the
domes t i c hor s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 1 . 7 . 3 . 1 . Infert i l i ty: commen t s o n termino logy • ...•.. 2 2 1 . 7 . 3 . 2 . Chromo s ome anoma l i e s i n the domestic
hor se . .. .. ...... ...... ............... .... .. . 26
CHAPTER II - Materials and Methods 28
2 . 1 . Subj ec t s •. ....•.......• •• •• ...... ..................• •. ..... .. 29 2 . 2 . Cytogene t i c techn i ques • . . . . . . • • . • . . . . • • • • • . . • . . . . . . . . • . . 29 2 . 2 . 1 . Leucocyte cu l tu r e s . . . . . . . . . . . . . • . • • • • . . . . . . . . . . . . . . . • 30
2 . 2 . 2 . Cel l cultu res other than l eucocyte cultures • • • • • • • . • 30 2 . 2 . 3 . Harvesting c e l l cultures and s l ide preparation . . . 30 2 . 2 . 4 . Staining . . . ... . . 30
2 . 2 . 5 . Sex- chromatin 30
2 . 2 . 6 . Evaluation of cul tured cel l s . . . • . . . 3 1 CHAPTER Ill - H- 1 1 , A fert i l e XY/XYY/?XO Mo saic/Chimaer;c S tallion 3 2 3 . 1 . Introduction . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3 . 2 . Materials and methods . . . 3 6 3 . 2 . 1 . Sources o f information . . . 36 3 . 2 . 2 . H - 1 1 . . . • . . . 36 3 . 2 . 2 . 1 . Description . . . • . . • • . . . 3 6 3 . 2 . 2 . 2 . General his tory . . . • • • • • � • . . . . • • . 36 3 . 2 . 2 . 3 . Reproduc t ive performance . . . : • . . . • • 37
3 . 2 . 2 . 4 . H- l l ' s s i re and dam • . . . • • • • • . • . . . • • • • • • • • . 38 3 . 2 . 3 . Relatives and progeny investigated • • . . . • • • • • • • . • • 38 3 . 2 . 4 . Cytogenetic materials and methods • • • • • • • . • . • . . • • . . • . 41
3 . 3 . Resu l ts of cytogenetic analysi s of H- 1 1 • • . . . • • . . . • • • • . . 44 3 . 4 . Extended cytogenetic s tudy of H- 1 1 : hi s relatives and
progeny . . . . . . . . . . • . • . . . • • . . . . . . . . . . . . . . . . . . . . . . . 50 3 . 5 . Di scussion . . . • . . . • . . . • . . . • . . . 58 3 . 5 . 1 . H - 1 1 ' s ferti l i ty . • . . . • . . . • . . . . . . . . . . . . . . . . . 5 8
3 . 5 . 2 . H - l l ' s XO f i l ly . . . • . . . 59 3 . 5 . 3 . The abnormal chromosome arrangements in cul tured
c e l l s of H- 1 1 ' s fami ly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 3 . 5 . 4 . H- l l ' s size and behaviour in view of hi s XYY line . . . • . 6 1 3 . 5 . 4 . 1 . H- l l ' s s1.ze . . . .. . . 6 1
3 . 5 . 4 . 2 . H- l l ' s behaviour . . . • . . . • • . . 63 3 . 5 . 5 . Possible manners of formation of H- l l ' s
mosai c /chimaeri sm . . . • . . . • • . . . . • • • • . . . . . . . . . . 64 3. 5 . 6 . Summary . . . • . . . • . . . • . . . . • . . . . • • • • . . . . . . . . . 65
v i i
CHAPTER IV - H-52, A Mare of Dis tinction 66
4 . 1 . Introduc tion ••• •• ... .. ••.....• .•• ••...•. ..... .....• .....•..• 67
4 . 2 . Material s and methods .• ...•... •.•• ... .•.• .....• •• .. ......... 69
4 . 2 . 1 . Description ... .• • •• ......• •.... .•.•• •••.........• ... 69
4 . 2 . 2 . History • • •.... ........• •..• ........•• ••..........•.. 69
4 . 2 . 3 . Cytogenet i c material s and methods ..... ...•.......•.. 69
4 . 2 . 3 . 1 . Ce l l cultures ..... ..•• .•.. . ............... 69
4 . 2 . 3 . 2 . Sex- chromatin investigations ........•..... 7 1 4 . 2 . 4 . Examinations and observations . . . 7 1 4 . 3 . Resu l t s . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4 . 3 . 1 . Resu l t s of cytogenetic investigations . . . 73
4 . 3 . 1 . 1 . Lymphocyte cu ltures . . . 86
4 . 3 . 1 . 2 . Cu l tured cel l s other than lymphocytes . . . 90
4 . 3 . 1 . 3 . S ex- chromatin s tudies . . . 96
4 . 3 . 2 . Reports of examinations and observations •..••• ...... 99
4 . 3 . 2 . 1 . Physical examinations of reproductive organs . . . 99
4 . 3 . 2 . 2 . General medical hi s tory at Massey Universi ty . . . 99
4 . 3 . 2 . 3 . Serum hormone analysis • ...•••• ...•••• ...• 101
4 . 3 . 3 . Post-mortem findings ...• ...• •...• •.• ....•.•• ••••• •. 102
4 . 3 . 4 . Observations on H- 52' s behaviour •••• .....•••• .•.••. 1 1 1 4 . 4 . Di scussion ....... ..... .. .•.... ........... •.... ............. 1 1 2 4 . 4 . 1 . Cytogenetic aspec t s of H-5 2 ' s mixoploidy 1 1 2 4 . 4 . 1 . 1 . The chromosome cons ti tution of H-52 • .•... 1 1 2 . 4 . 4 . 1 . 2 . The po ssible zygote/ embryonic chromosome
consti tution of H - 5 2 . . . 1 13 4 . 4 . 1 . 3 . Po ss ible predi sposing fac tors to H-5 2 ' s
mixoploidy . . . 1 1 7 4 . 4 . 2 . Phys ical and behavioural aspect s o f H-53 ' s
mixoploidy . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . • • . . 1 1 7 4 . 4 . 3 . Summary .. . .. .. .. . . .. . ... ...•.. . . . . . . . . . . . .. . . ... . .. 1 21
CHAPTER V - Two 64,XX/ 65,XXY Mosaic / Chimaeric Mares 1 22 5 . 1 . Introduction ...•• . ... .. •••. .... •.• ••.••..... .••..•••.•• 1 23
5 . 2 . Materia l s and methods •• •• . •.• •• ••••. .. . .•••• .•. .. . ...•. 1 24
5 . 2.1 . Sources of information . . .. . . . .. . .•. .. .. ... •...•. 1 24 5 . 2 . 2 • H- 46 • . . . . . . . . • . . . . . . . . . . . . • . • . . . . . . • . . . • . . . . 1 2 4
5 .2 . 2.1 . Description . .. • .. . . . .. . . . .. . ... . . ... .. 1 24 5 . 2 . 2 . 2 . Feminine structures . . .. . . . . .. . . . .. .. . . 1 24 5.2 . 2 . 3 . Reproduc tive hi s tory .. . ... . . . .. . . . 1 24
5 .2 . 2 . 4 . Behaviour • .. .• . .. . . . ... .. . . . • ... . .. . .. 1 27 5 .2.3. H - 47 ... . . .. . . . .. . . .. . . .. .. . . .. .. .... . . .. .. . 1 27 5 . 2 . 3 . 1 . Description • . . . ..... ... . . ....•• ... .. .. 1 27 5.2 . 3 . 2.
5 . 2.3 . 3 . 5 . 2.3.4 .
General hi story . ......• ••.. . .••.••... . 1 27 Reproductive his tory •..• ••. .. . ...••••• 1 29 Behaviour . . . . . 1 3 1 5 .2.4. Cytogeneti c material s and methods •• ..•• ••••••• • • 1 3 1 5 . 2 . 4 . 1 . H- 46 . .. .. ... •.. .... ........... ........ 1 3 1 5.2.4 . 2. H- 47 • •• •• ....•• ••... . . ••• • •• ..•.•• ..•• 13 1 5 .2 . 4 . 3 . Evaluation of cel l s •••• •••.•.••.•... .. 1 3 1 5 .3 . Results •• ..... ..• •. .. .. ...• .. ... . .. .•. . . .. .•. ..•. •....• 1 3 2
5 . 4 .
5 . 3 . 1 . H-46 5 .3 . 2 . H-47
Discussion .... . . . ... . ... . . . .... ... . . .. . ... . . . .. . 1 3 2 1 3 6 140 5 .4 . 1 . Errors of development and H - 46 and H- 47 . ... 144
5 .. 4.2. Summary .. ... .. • . . .. . .... •.... . . . . . . . .... ..... .. . 1 47
CHAPTER VI - H- 27, An Enigma 1 48
6 . 1 . Introduction • .•.. • . .. . ..•. ..• .• .•• •..... •.. ..... .•..... 1 49 6.2 . Materials and methods • ..• •.•• ......•••. .•. ....••....... 1 5 1 6.2.1 . H- 27 . . ... . ... .. ... .... . . ... .. .. ... .. ... . 1 5 1
6 . 2 . 1 . 1 . Descri p tion .• ..•••••••. .... • •••..•...• 1 5 1 6 . 2 . 1 . 2 . General information •..•• •••••.•••..•.• 1 5 1
i x
6 . 3 .
6 . 2 . 1 . 3 . Pre- admi ssion history • . . . . ••. •..• • . . . 1 5 1 6 . 2 . 1 . 4 . History at Massey University ... .......•.. 1 5 1 6 . 2 . 1 . 5 . Behavi our . . . 1 5 2
6 . 2 . 1 . 6 . Pos t-mortem report ....• .•.• ...... ...• ... 1 5 2 6 . 2 . 2 . Cytogeneti c material s and methods •..•••....... ..•. 1 55
6 . 2 . 2 . 1 . Cu ltured cel ls .....• .•• .•........•• •.... 1 55
6 . 2 . 2 . 2 . S ex- chromatin investigations ...•.•••• •.• 1 5 6 6 . 2 . 2 , 3 . Lymphocyte transformation test .•..••• .•. 1 5 6
Resu l t s 1 5 7
6 . 3 . 1 . Resu l t s of cytogeneti c investigations -.... .. . . 1 5 7 6 . 3 . 1 . 1 . Cultured cel l s ••• .••.•• ...•• •. • ....•.. .. 1 5 7
6 . 3 . 1 . 2 . Sex- chromatin invest igations . .. . . .... .. 1 67 6 . 3 . 2 . Lymphocyte pro l iferation test .•.. .......•.....••• . 1 69 6 . 4 . Di scussion . . . .. . . 1 7 2
6 . 4 . 1 . Cytogeneti c abnormality .. . ...... . . .. . . ... . . . 1 7 2 6 . 4 . 2 . Some impli cations of the fai lure of lymphocyte
cu ltures ... . . ... . . ..... . . . . . . ... . . . . . . . 1 73 6.4.3 . Po s s ible impli cations of mitotic instabi lity in
c e l l cu l tures . ..•...... ....•.•...... ••........•• .•. 1 74 6 . 4 . 4 . Inferti l i ty
6 . 4 . 5 . Summary . . ... ... . ... . . . .. . . .. . . . .. . . .. . .
CHAPTER VII - General Discussion
7 . 1 . Preval ence of the mo saic /chimaeric condi tion in
1 7 6 1 7 7 1 79
�- .£.· cabal lus . . . . . . . . . . . . . . . 180 7 . 2 . Possibl e predi spo sing factors to the mosaic / chimaeri c
condi tion and the horses in this study •••. ••••••.•••••• •..•• 1 8 2
7 . 2 . 1 . Parental age effect 1 8 2
7 . 2 . 2 . Birth order and dam' s obstetric hi s tory •.• •••• ••.• • 183 7 . 2 . 3 . Possible inf luences of metabol i sm and physio logy ••• . 1 8 4 7 . 2 . 4 . Inbreeding and relationships between the mosai c /
chimaeras i n thi s study ...•...•• .••••• ...••.. .•.... 185
7 . 2 . 5 . Genetic predi sposi tion to masked karyotype
change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . 1 8 6 7 . 3 . Mosai c / chimaeric individual s and breed sub-ferti lity . . . 1 8 8
7 . 3 . 1 . Mosaic/ chimaerism and fert i l i ty . . ..........•..•.• 188 7 .4 . Cytogenetics and the Domestic Horse . . .. . . .. . .. . . 1 9 1
Appendix I - The 1978 Pi lot Survey
Appendix ll - Unpubl i shed XO Mares Di s covered in New Zealand and Other Horses Inves tigated for which there
193
was a Sugges ti on of a Cytogenetic Anomaly •••••••• 200 Appendix Ill - Anci l lary Information to Chapter IV
A . H - 5 2 1s Dimen sions
B . H - 5 2 Pos t - Mortem • ...• ..•• ......•••• · �.. .. 204 C . Estimates of Pr imordia l Ce l l Pool -Size . . . 207 Appendix IV - H- 27 Post Mortem . . . . . . . . . . . . . . . . . . . . . . . . . . 209 Appendix V - Materials
A. Supp l i es 2 1 1
B . Tissue cul ture materia l s .•............... 2 1 2
c.
D .
Cel l cu lture media Staining solutions
2 1 4 2 1 4 E . E qui pment . . . . . . . . . . . • • . . . . . . . . . 2 1 5 Appendix V I - Methods
I . Leucocyte culture system • . . . . • • • . • . . • . . • . . 21 6 1 1 . Cel l culture techni ques • • . • . . • • • . . . • • . . . • . . • 2 1 7 I II . Slide preparation • • . • • • • . . . . • . . • • • • . . . • . . . 2 1 9 IV . Sex- chromatin • • • • • . • . . • • • • . • . . . • . . • • • . . • 220 V . Differential staining . • • • . • . . • • • • • • • . . . . • • . . 220 VI . Photographic methods .•..• .•• •.....••••••• . • • 2 2 1 VII . Lymphocyte transfo r ma. tion test . • • . • . • • • • . • . • 2 2 2
-
B i b l i ography • • • . • . . . . • • • • . • . • . . . . • • • • • . . . . . . . . • • . • • • . • • . • • • 2 24 xi
