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Factors contributing to the maintenance and activation of corynebacterial pseudotuberculosis in the laboratory… Bruce, David Lorne 1969

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FACTORS CONTRIBUTING TO THE MAINTENANCE AND ACTIVATION OF CORYNEBACTERIAL PSEUDOTUBERCULOSIS IN THE LABORATORY MOUSE by DAVID LORNE BRUCE B.Sc.  (Honors), U n i v e r s i t y of B r i t i s h Columbia,  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor o f Philosophy i n the Department of Microbiology  We accept t h i s t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1969  1966  In p r e s e n t i n g an  this  thesis  advanced degree at  the  Library  I further for  shall  the  his  of  this  written  agree that  University  of  permission  representatives.  be  available  A p r i l 28,  g r a n t e d by  gain  Microbiology  1969  for  for extensive  permission.  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  British  the  It i s understood  thes,is f o r f i n a n c i a l  Department of  Date  f u l f i l m e n t of  make i t f r e e l y  s c h o l a r l y p u r p o s e s may  by  in p a r t i a l  Columbia  shall  requirements  Columbia,  Head o f my  be  I agree  r e f e r e n c e and copying of  that  not  the  that  Study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  i ABSTRACT C o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s , a murine d i s e a s e caused by Corynebacterlum k u t s c h e r l . o c c a s i o n a l l y i n numbers of the departmental  developed  s t r a i n of random-bred Swiss  white mice 3 to 5 weeks f o l l o w i n g i n t r a p e r i t o n e a l of  the Rauscher leukemia v i r u s .  extremely d i f f i c u l t animals, was  V i r u l e n t C. k u t s c h e r l ,  to I s o l a t e from a p p a r e n t l y h e a l t h y  f r e q u e n t l y c u l t u r e d from the organs of animals  p r e s e n t i n g symptoms of the Rauscher pseudotuberculosis. mental  injection  d i s e a s e without a c t i v e  R e p r e s e n t a t i v e groups of the d e p a r t -  s t r a i n showed u n i f o r m l y h i g h r e s i s t a n c e to i n t r a v e n o u s  i n j e c t i o n of v i r u l e n t C. k u t s c h e r l i n comparison  to imported  pathogen-free s t r a i n s , y e t succumbed to p s e u d o t u b e r c u l o s i s i n l a r g e numbers f o l l o w i n g c o r t i s o n e treatment; t h e r e f o r e the e n t i r e colony was  assumed to be l a t e n t l y i n f e c t e d by t h i s  organism. Two to  s t r a i n s of C. k u t s c h e r l were I s o l a t e d and compared  s t r a i n s ATCC 156?? and ATCC  presumed non-pathogenic  15678.  The l a t t e r s t r a i n , a  v a r i a n t of the former, was  compared  by c e l l w a l l sugar and d e o x y r i b o n u c l e i c a c i d base a n a l y s i s to  the pathogenic s t r a i n .  methods to f i n d of  not p o s s i b l e by these  support f o r the v a r i a n t h y p o t h e s i s .  the l a t e n t s t a t e from the departmental  f r e e s t r a i n of mice was of  I t was  attempted  s t r a i n to a  demonstrated.  pathogen  by i n j e c t i o n of homogenates  v i s c e r a l organs, but t r a n s f e r of l a t e n c y was  fully  Transfer  not success-  The  sera of l a t e n t l y i n f e c t e d mice s t i m u l a t e d  in  v i v o p h a g o c y t o s i s of C. k u t s c h e r l by mouse p e r i t o n e a l macrophage, but  i t was  not p o s s i b l e to demonstrate c i r c u l a t i n g  C. k u t s c h e r l s p e c i f i c antibody i n non-immunized animals. Immunized normal mice of a l l s t r a i n s d e v e l o p e d  high  titers  of s p e c i f i c c i r c u l a t i n g antibody f o l l o w i n g i n j e c t i o n s of e i t h e r heat k i l l e d  C. k u t s c h e r l c e l l s or v i r u l e n t organisms  i n numbers too loi\r to i n i t i a t e i n f e c t i o n . body was and  Circulating anti-  demonstrated by p a s s i v e b a c t e r i a l hemagglutination  by i n d i r e c t f l u o r e s c e n t antibody s t a i n i n g .  m u n i z a t i o n was  When im-  attempted d u r i n g e a r l y Rauscher d i s e a s e ,  C. k u t s c h e r l antibody p r o d u c t i o n Clearance s t u d i e s w i t h  was  inhibited.  l a b e l e d C.  showed that the organism i s c l e a r e d with  kutscherl  exceptional  r a p i d i t y by the r e t i c u l o e n d o t h e l i a l system o f l a t e n t l y i n f e c t e d mice, pathogen f r e e mice and mice w i t h e a r l y Rauscher d i s e a s e . slightly  The  i n the above  clearance order.  constant  decreased  iii TABLE OF CONTENTS Page Introduction  1  M a t e r i a l s and Methods  5  Animals  5  Virus t i t r a t i o n  5  I n d u c t i o n of c o r y n e b a c t e r i a l pseudot u b e r c u l o s i s w i t h RLV  6  R e t i c u l o c y t e counts  8  Microorganisms and c u l t i v a t i o n  9  Determination of deoxyribonucleic (DNA)  acid 9  base r a t i o s  I d e n t i f i c a t i o n of c e l l w a l l sugars Examination of C. k u t s c h e r l  10  culture  supernatant f o r t o x i c p r o p e r t i e s I s o l a t i o n procedures f o r C. k u t s c h e r l  11 13 1*J-  Virulence Latency t r a n s f e r attempts  15  Cortisone  17  administration  Immunization  o f mice  17  Macrophage s t u d i e s  19  Serology  21  Clearance s t u d i e s  23  E l e c t r o n microscopy  2k  iv Page 26  Results  26  Virus t i t r a t i o n Rauscher leukemia v i r u s induced  coryne-  b a c t e r i a l pseudotuberculosis  31  R e t i c u l o c y t e counts  33  Microorganisms and c u l t i v a t i o n  37  Pathogenicity determination  44  I s o l a t i o n of C. k u t s c h e r l  53  Latency t r a n s f e r attempts  54  Cortisone administration  56  Macrophage  60  studies  Immunization and serology  64  Clearance  68  studies  E l e c t r o n microscopy  70 73  Discussion Rauscher leukemia v i r u s (RLV)  73  Corynebacterial pseudotuberculosis  75  Immunodepression  81  Phagocytosis  86  Clearance  i n vitro  90  studies  Summary and c o n c l u s i o n s  94 105  Bibliography  >  V Page Appendices I II III IV  113  C a l c u l a t i o n of RLV ID^Q  113  C a l c u l a t i o n of DNA Base R a t i o s  Ilk  D e t e r m i n a t i o n of P h a g o c y t i c I n d i c e s  116  C a l c u l a t i o n of Average Constant K f o r Clearance o f p32 Labeled C. k u t s c h e r l  118  vl  LIST OF TABLES Page I  RLV i n j e c t i o n and i n c i d e n c e o f pseudotuberculosis  II III IV V VI  28 35  R e t i c u l o c y t e counts B i o c h e m i c a l r e a c t i o n s and other p r o p e r t i e s of C. k u t s c h e r l s t r a i n s S u r v i v a l o f WSW mice i n j e c t e d w i t h v a r i o u s C. k u t s c h e r l s t r a i n s  45  S u r v i v a l o f d i f f e r e n t mouse s t r a i n s i n j e c t e d w i t h C. k u t s c h e r l ATCC 1 5 6 7 7  46  S u r v i v a l of d i f f e r e n t mouse  strains  i n j e c t e d w i t h C. k u t s c h e r l s t r a i n Wl VII VIII IX  39  47  E f f e c t of c o r t i s o n e a d m i n i s t r a t i o n  58  In v i t r o phagocytosis  62  Passive  67  hemagglutination t i t e r s  vii  LIST OF FIGURES Page 1.  Rauscher v i r u s I D ^ Q d e t e r m i n a t i o n  2.  Corynebacterial pseudotuberculosis a mouse  3.  Spleens o f RLV I n f e c t e d and normal mice  4. R e t i c u l o c y t e s i n p e r i p h e r a l 5.  27 in  blood  30 30 34  P r o g r e s s i v e r e t i c u l o c y t o s i s i n Rauscher disease  36 42  6.  Gram s t a i n s o f C. k u t s c h e r l s t r a i n s  7.  C e l l w a l l sugar chromatography  ^3  8. C o r y n e b a c t e r i a l abscess i n peritoneum 9. C o r y n e b a c t e r i a l abscess i n kidney  cortex  10.  C. k u t s c h e r i i n crushed l i v e r nodule  11.  C. k u t s c h e r i i n kidney abscess  12.  I n f i l t r a t i o n of l e s i o n by lymphocytes  13.  Cortisone  14.  C e l l s i n mucoid a s c i t e s f l u i d  15.  Mouse p e r i t o n e a l macrophages i n c u l t u r e  16.  Immunofluorescent s t a i n i n g  17.  Clearance  induced  pseudotuberculosis  o f C. k u t s c h e r i by RES  1 8 . E l e c t r o n micrographs of C. k u t s c h e r l 1 9 . R e s i s t a n c e to s u p e r i n f e c t i o n and a c t i v a t i o n of latency 2 0 . I n f e c t i o n , l a t e n c y and a c t i v e d i s e a s e  48 48 49 49  50 56 57 63 66 67 71 103 104  ACKNOWLEDGEMENT I am p l e a s e d t o acknowledge my indebtedness t o Dr. J . E. Bismanis f o r h i s t h o u g h t f u l c o n s i d e r a t i o n and help w i t h the problems which I have encountered d u r i n g t h i s study, and t o express my s i n c e r e apprec i a t i o n f o r h i s guidance, h i s i n s t r u c t i o n and h i s friendship. I wish a l s o t o thank a l l  the f a c u l t y  members  of t h e Department o f M i c r o b i o l o g y , and Dr. W. L. Dunn of t h e Department o f Pathology, f o r t h e i r k i n d and w i l l i n g a s s i s t a n c e and t h e i r h e l p f u l a d v i c e .  1  INTRODUCTION The  r e l a t i o n s h i p between the m i c r o b i a l p a r a s i t e  and  i t s animal host has been studied.most e x t e n s i v e l y d u r i n g disease  state.  the  However, many h o s t - p a r a s i t e r e l a t i o n s h i p s  i n v o l v e the prolonged p e r s i s t e n c e of microorganisms without p r o d u c t i o n of c l i n i c a l the l a t t e r s t a t e may Salmonella  illness.  Depending on the organism,  be r e a d i l y demonstrated, as  with  t y p h i , o r almost Impossible to d i s c o v e r ,  R i c k e t t s i a prowazekll  (26).  Various  terms have been used  to d e s c r i b e e s s e n t i a l l y s i m i l a r s i t u a t i o n s , and the c a r r i e r s t a t e o r inapparent  e.g.,  they i n c l u d e  (latent, subclinical  or  dormant) i n f e c t i o n s , a l l c o n d i t i o n s where the i n f e c t i o u s agent has present  so m i l d an e f f e c t t h a t even though i n f e c t i o n be  and  i s i d e n t i f i a b l e by l a b o r a t o r y means, i t i s  undetected c l i n i c a l l y  (2).  For t h i s study, the l a b o r a t o r y mouse was  s e l e c t e d as  a model f o r the study of some of the mechanisms which e i t h e r a l l o w the establishment  may  and p e r s i s t e n c e of a l a t e n t  i n f e c t i o n o r p r e c i p i t a t e i t s development i n t o a c t i v e d i s e a s e . A common mouse pathogen w i t h which e n t i r e c o l o n i e s of mice have been shown to be l a t e n t l y i n f e c t e d i s Corynebacterium kutscherl  (3*0.  T h i s microorganism i s the c a u s a t i v e agent  of murine c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s ,  a d i s e a s e which  i n i t s a c t i v e form i s c h a r a c t e r i z e d by r u f f l e d f u r , a waddling g a i t , and  the presence of gross,  caseous abscesses  2 i n o r a s s o c i a t e d w i t h the l u n g s o r other (3,  11,  disease  3 3 , ^ 3 , k7).  Rats a r e a l s o s u s c e p t i b l e to the  (3, ^3, ^9, 79).  The organism i s i s o l a t e d from  i n d i v i d u a l mice i n a p p a r e n t l y extreme d i f f i c u l t y  i n t e r n a l organs  healthy  c o l o n i e s only w i t h  ( 7 ) but i t has been shown i n numerous  i n s t a n c e s t h a t the d i s e a s e  can be a c t i v a t e d i n r e p r e s e n t a t i v e  groups- of the same c o l o n i e s  i f these groups a r e subjected t o  conditions of p h y s i o l o g i c a l stress.  Reported  conditions  have i n c l u d e d  X - i r r a d i a t i o n ( 7 2 ) , v i t a m i n d e f i c i e n c y (82),  salmonellosis  (80), i n f e c t i o u s e c t r o m e l i a  t r a t i o n of cortisone  ( 3 , Jk, ^ 9 ) .  f o l l o w i n g the o b s e r v a t i o n  (48) and adminis-  T h i s study was undertaken  t h a t numbers of our departmental  s t r a i n of random-bred Swiss white mice developed a c t i v e corynebacterial pseudotuberculosis Rauscher leukemia v i r u s The  f o l l o w i n g i n f e c t i o n by the  (15).  Rauscher v i r u s i s a potent leukemogenic v i r u s  t h a t induces a dual type o f d i s e a s e . disease  Involves  The f i r s t  stage o f the  a p r o l i f e r a t i o n of predominantly  erythro-  c y t i c and l e u k o c y t i c elements, b e g i n n i n g as e a r l y as 7 days after parenteral  inoculation.  Splenomegaly u s u a l l y develops  w i t h i n 15 days and a high percentage of the animals d i e w i t h i n 25 t o 35 days.  In the m a j o r i t y  of the mice s u r v i v i n g  the e r y t h r o c y t o p o i e s l s , a t r a n s p l a n t a b l e lymphocytic leukemia develops between 30 and ^5 days a f t e r i n o c u l a t i o n ( 6 5 ) . The  i n t e r a c t i o n s i n v o l v e d i n t h i s study i n c l u d e :  3 1. Those defense mechanisms of the murine host which tend to suppress a c t i v e 2. The  a b i l i t y of the  survive  pseudotuberculosis.  "dormant" C. k u t s c h e r l  to  i n the l a t e n t l y i n f e c t e d host and i t s  tendency to develop i n t o a c t i v e d i s e a s e  in a  more f a v o r a b l e p h y s i o l o g i c a l environment. 3. Those p a t h o l o g i c a l e f f e c t s of the Rauscher v i r u s i n f e c t i o n which tend to counteract  those  defense mechanisms of the l a t e n t l y i n f e c t e d host which are m a i n t a i n i n g  the  C.  kutscheri  i n a dormant s t a t e . Because d i f f e r e n t s t r a i n s of mice vary g r e a t l y i n t h e i r r e s i s t a n c e to C. k u t s c h e r l  i n f e c t i o n (62), both s u s c e p t i b l e  r e s i s t a n t s t r a i n s were used, and  s e v e r a l f a c t o r s which might  e x p l a i n these s t r a i n d i f f e r e n c e s were examined. cluded  p o s s i b l e f a c t o r s i n the  and  These i n -  "normal" sera of r e s i s t a n t  s t r a i n s of mice, lmmunospecific or otherwise, which might f a c i l i t a t e p h a g o c y t o s i s of C. k u t s c h e r l by p e r i t o n e a l macrophage; a comparison of the antibody response to C. antigen  by r e s i s t a n t and  s u s c e p t i b l e mice; and  the r e l a t i v e r a t e s of c l e a r a n c e system (RES)  of r e s i s t a n t and  susceptible  i n the  from l a t e n t l y i n f e c t e d mice excepting  s t r a i n s of mice  under the  induced d u r i n g development of the Rauscher d i f f i c u l t y reported  a study of  by the r e t i c u l o e n d o t h e l i a l  under d i f f e r e n t immune c o n d i t i o n s and  The  kutscheri  conditions  disease.  i s o l a t i o n of C.  kutscherl  after "activation"  4 procedures (7, 18, 6 2 ) and the f a c t that o n l y one worker has , reported  any success i n d u p l i c a t i n g t h e pathology o f the  n a t u r a l l y o c c u r r i n g d i s e a s e by a d m i n i s t r a t i o n o f v i r u l e n t C. k u t s c h e r i  ( 6 3 ) has l e f t  some doubt as t o the s t a t e i n  which l a t e n t l y i n f e c t i n g organisms e x i s t , and has l e d Fauve et  a l ( 3 4 ) t o suggest C. k u t s c h e r l ATCC 15678 (an a v i r u l e n t ,  c o c c o i d organism) as an a l t e r n a t e b a c t e r i a l form. v i t r o transformations  have y e t been r e p o r t e d .  No i n  Because the  e x i s t e n c e o f such a v a r i a n t would s t r o n g l y encourage the d i r e c t i o n o f t h i s i n v e s t i g a t i o n toward a study o f the f a c t o r s which c o u l d I n i t i a t e r a d i c a l change i n the i n f e c t i n g microorganism, a comparative study o f the c e l l virulent  composition of the  C. k u t s c h e r l ATCC 1 5 6 7 ? and the proposed non-  pathogenic v a r i a n t , C. k u t s c h e r l ATCC 1 5 6 7 8 , was undertaken (16).  Also  i n v e s t i g a t e d was the p o s s i b i l i t y of t o x i n s  pathogenic f o r mice b e i n g produced by C. k u t s c h e r l .  The  absence of any t o x i n s would be expected to enhance the p o s s i b i l i t y o f p e r s i s t e n c e o f the organism f o r l o n g  periods  of time without o b s e r v a b l e e f f e c t on the host,  facili-  t a t i n g increase  thus  i n the number o f c a r r i e r s and i n f e c t i o n of  e n t i r e c o l o n i e s o f animals.  5 M a t e r i a l s and Methods Animals.  The departmental stock o f random bred Swiss  white mice, designated as WSW,  c o n t r i b u t e d most o f the mice  which were used i n t h i s study because i t was i n t h i s  colony  that outbreaks o f c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s , the d i s e a s e under study, were f i r s t  noted.  The o t h e r s t r a i n s of  mice used were A/J, purchased from Jackson L a b o r a t o r i e s , Bar Harbor, Maine; and CF-^SPF), from Carworth Farms, New C i t y , N.Y.  The l a t t e r two s t r a i n s were r e p o r t e d by Fauve e t a l (34)  to be f r e e o f l a t e n t c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s (C. k u t s c h e r l f r e e ) as r e c e i v e d from the designated  suppliers.  The use of both C. k u t s c h e r l f r e e and l a t e n t l y I n f e c t e d mice was r e q u i r e d to enable comparisons to be made between the s t r a i n s which might o f f e r i n s i g h t i n t o which f a c t o r s  contri-  bute s t r o n g l y to the maintenance o r a c t i v a t i o n o f coryneb a c t e r i a l p s e u d o t u b e r c u l o s i s and which do not. The imported, C. k u t s c h e r l f r e e s t r a i n s were housed i n Isocages (Lab Cages Inc., Hackensack,  N.J.) i n order to m i n i -  mize o p p o r t u n i t i e s f o r c r o s s - i n f e c t i o n d u r i n g the course of experiments i n which they were used. housing were not s t e r i l i z e d .  However, t h e i r food or  A l l mice were f e d P u r i n a Rat  Chow ( R a l s t o n P u r i n a Co.) and water ad l i b i t u m . Virus t i t r a t i o n .  Murine leukemia v i r u s ,  Rauscher  s t r a i n (RLV) was s u p p l i e d by Flow L a b o r a t o r i e s ,  Rockvllle,  Maryland, i n a f r o z e n c o n d i t i o n , and was t h e r e a f t e r maintained at -80C u n t i l use.  I n an experiment u n r e l a t e d to t h i s study,  6 the ID^Q  °f t h i s v i r u s shipment f o r WSW  sub-cutaneous i n j e c t i o n of 0.1  mice was  titrated  ml each of a s e r i e s of f i v e  1 0 - f o l d d i l u t i o n s of v i r u s i n Hanks' balanced s a l t A t o t a l of 11?  solution.  neonatal mice were used i n t h i s d e t e r m i n a t i o n  (Table I, the second to the f i f t h group, i n c l u s i v e ) . megaly, a f t e r 21 days, o r p r i o r death, was dence of Rauscher d i s e a s e I t was  Spleno-  accepted as  evi-  (65).  the o b s e r v a t i o n of unexpected  these mice, from which G. k u t s c h e r l was  l e s i o n s i n two  subsequently  f i e d , t h a t I n i t i a t e d the present study.  RLV  used  of  identi-  The t i t r a t i o n d a t a  are a l s o very r e l e v a n t to the p r e s e n t study because t h i s of v i r u s was  by  i n a l l the f o l l o w i n g experiments  i n d u c t i o n of p s e u d o t u b e r c u l o s i s was  stock  i n which  attempted.  The  ti-  t r a t i o n r e s u l t s are i n t e r p r e t e d g r a p h i c a l l y i n F i g . 1 and i n the accompanying d i s c u s s i o n , and the c a l c u l a t i o n of the  ID^Q  set f o r t h i n Appendix I. I n d u c t i o n of c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s w i t h RLV.  T h i s s e r i e s o f experiments  was  i n i t i a t e d to  determine  whether the occurrence of c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s i n the course of RLV phenomenon.  Two  i n f e c t i o n (see above) was  cases i n a group of 117  (46 w i t h splenomegaly) c o u l d be merely  a reproducible  animals  injected  coincidental.  It  was  noted t h a t i n s t u d i e s of c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s r e p o r t e d by o t h e r workers, a d u l t mice were g e n e r a l l y used. Fauve et a l (3*0  r e p o r t e d t h a t age appeared  to be of no  sig-  n i f i c a n c e i n the a c t i v a t i o n of l a t e n t c o r y n e b a c t e r i a l pseudot u b e r c u l o s i s by a d m i n i s t r a t i o n of c o r t i s o n e .  On the o t h e r  7 hand, a d u l t mice are f a r l e s s s u s c e p t i b l e to RLV than are neonatal o r weanling mice ( 2 7 , As a compromise between the two  infection  65). extremes, i t  was  d e c i d e d to use weanling mice ( 4 - 5 weeks o l d ) i n the 1 1 4 (72  study, and  males and  initial  4 2 females) were used.  Large  doses of v i r u s were i n j e c t e d (50 or 1 0 0 I D ^ Q ) because of age  and  r e l a t i v e l y high r e s i s t a n c e of these mice.  periment was  The  staggered over a twelve week p e r i o d to  time f o r c a r r y i n g out gross i n s p e c t i o n s , making and touch p r e p a r a t i o n s  of lungs,  l i v e r s , kidneys and  the  ex-  allow staining  spleens,  and  attempting c u l t u r e of b a c t e r i a from these organs. Using the c a r r i e d out  same procedures, f u r t h e r experiments were  i n which a d u l t mice ( 8 - 1 2 weeks o l d ) were used. 72 WSW  These i n c l u d e d  CF-^(SPF) males and  mice ( 4 8 males and  2 0 A/J males.  The  2 4 females),  l a t t e r two  i n c l u d e d as G. k u t s c h e r l f r e e c o n t r o l s . ( 1 8 males and  t i t r a t e d v i r u s was  a concentration  of 1 0 0 I D ^  intraperitoneally. the  mice  age,  the  I n j e c t e d mice.  i n j e c t e d i n Hanks' s o l u t i o n at P ^ ml, E  0  to  0.5  o r 1 ml being i n j e c t e d  I n j e c t e d mice were caged i n groups of 6  course of the  of mice, sex,  36 a d u l t WSW  these were subjected  same i n s p e c t i o n procedures as RLV  during  s t r a i n s were  1 8 females) were a l s o i n c l u d e d as v i r u s - f r e e  l a t e n t l y i n f e c t e d c o n t r o l s , and  The  12  experiment.  The  d e t a i l s of numbers  dosage, i n i t i a t i o n of RLV  infection, i n c i -  dence of p s e u d o t u b e r c u l o s i s and  d e t e c t i o n of C.  kutscherl  organisms i n touch p r e p a r a t i o n s  of organs are i n c l u d e d  in  8 Table I and  c o n s i d e r e d i n the accompanying d i s c u s s i o n .  p r e p a r a t i o n s of organs were a i r - d r i e d , methanol f i x e d Giemsa s t a i n e d .  The  R e t i c u l o c y t e counts.  (27,  f o r C. k u t s c h e r i .  Splenomegaly i s the g e n e r a l l y  c r i t e r i o n f o r determining  65),  and  i n severe  pable w i t h i n 21 days. pseudotuberculosis,  infections,  Rauscher d i s e a s e spleens may  i t was  found  s p l e e n o r other organs of RLV days a f t e r i n f e c t i o n .  infection  become p a l -  In t h i s study of l a t e n t c o r y n e b a c t e r i a l t h a t f o c i of a c t i v e coryne-  b a c t e r i a l i n f e c t i o n were sometimes present  critical  and  c u l t u r e of b a c t e r i a from organs i s  d e s c r i b e d i n I s o l a t i o n procedures  accepted  Touch  liver,  i n f e c t e d mice as e a r l y as  T h i s 21 day  i n the pathogenesis  i n the  21  interval i s therefore  of p s e u d o t u b e r c u l o s i s ,  and  s t u d i e s i n immunodepression were t h e r e f o r e begun e a r l y i n this period.  However, i t was  necessary  to e s t a b l i s h whether  each immunized mouse had a c t u a l l y developed b e f o r e the antibody cance.  Rauscher d i s e a s e  t i t e r of a g i v e n serum had any  signifi-  With most mice, splenomegaly could be determined only  a f t e r 14-21  days, and  then with c e r t a i n t y o n l y a f t e r  sacrifice.  R e t i c u l o c y t o s i s , which f r e q u e n t l y occurs w i t h i n 7 days a f t e r RLV  infection,  i s the f i r s t d e t e c t a b l e abnormality  p e r i p h e r a l blood  (76)  i n j u r y to the mouse. to v e r i f y RLV  and  i n the  can be r e l i a b l y determined  A r e t i c u l o c y t e count was  without  t h e r e f o r e used  i n f e c t i o n d u r i n g the course of the  Immunization  programmes. Mice on which r e t i c u l o c y t e counts were to be determined  9  were r e s t r a i n e d and t h e t a i l s swabbed w i t h 70$ a l c o h o l . lateral and  t a i l v e i n was p r i c k e d with a No. 2k hypodermic needle  0.005  ml of blood taken by c a p i l l a r y p i p e t t e .  stained with b r i l l i a n t Wright's  C e l l s were  c r e s y l blue and c o u n t e r s t a i n e d with  s t a i n by the method of Osgood and Wllhelm  l e a s t 1,000  cells  were counted  Microorganisms k u t s c h e r i ATCC  15677  C. H. Pierce-Chase.  15678  A  At  i n each p r e p a r a t i o n .  and C u l t i v a t i o n . and ATCC  (6l).  15678  Corynebacterlum  were o b t a i n e d from Dr.  A second c u l t u r e of C. k u t s c h e r l ATCC  was o b t a i n e d from the American Type C u l t u r e C o l l e c t i o n .  Organisms d e s i g n a t e d C. k u t s c h e r l s t r a i n s W-l and W-2 I s o l a t e d i n our own l a b o r a t o r y . i n the n e c r o t i c l i v e r  of a WSW  as a weanling w i t h RLV.  C. k u t s c h e r l W-l was found  mouse which had been i n f e c t e d  C. k u t s c h e r l W-2  an abscess which appeared  were  was i s o l a t e d  i n the mesenteric node of a  from WSW  mouse which had been i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 10 mg of h y d r o c o r t i s o n e a c e t a t e 21 days p r e v i o u s l y . a departmental  strain.  E. c o l l used  was  C o r y n e b a c t e r i a l s p e c i e s were r o u t i n e l y  maintained on v e a l i n f u s i o n b r o t h agar o r blood agar.  Batch  c u l t u r e s were grown without a e r a t i o n i n v e a l i n f u s i o n b r o t h . D i f c o p r o d u c t s were used i n media p r e p a r a t i o n . Determination of D e o x y r i b o n u c l e i c A c i d Ratios.  Microorganisms  base composition. ATCC  15678  (DMA) Base  possess DNAs t h a t v a r y widely i n  The DNAs o f C. k u t s c h e r l ATCC  15677  and  were t h e r e f o r e compared as p a r t of a study of the  10  two  s t r a i n s , one of which (ATCC 1 5 6 7 7 ) i s pathogenic f o r (34,  mice, and the o t h e r a v i r u l e n t  62).  Because both of  these s t r a i n s are r e f r a c t o r y to l y s i s by e i t h e r lysozyme o r detergent treatment, harvested, washed c e l l s were d i s r u p t e d (51).  by an a l t e r n a t i v e method which i s d e s c r i b e d by Marmur 2 g wet weight  of c e l l s were ground f o r 10 min w i t h an equal  weight of g l a s s beads ( S u p e r b r i t e type #133, and Manufacturing mortar.  Minnesota  Mining  Company) w i t h s a l i n e EDTA i n a p r e - c o o l e d  Ten volumes of c o l d s a l i n e EDTA c o n t a i n i n g 2% sodium  l a u r y l s u l f a t e were added and the suspension c e n t r i f u g e d . The DNA  e x t r a c t i o n procedure  subsequently used on the super-  natant i s d e s c r i b e d i n d e t a i l by Marmur ( 5 1 ) .  Briefly,  the  c e l l d e b r i s and p r o t e i n are removed by d e n a t u r a t i o n (detergent and chloroform) and c e n t r i f u g a t i o n , the HNA and the DNA  removed by RNase  p r e c i p i t a t e d w i t h 95% e t h y l a l c o h o l .  base r a t i o of each v a r i e t y was  The  estimated from t h e i r r e s p e c t i v e  buoyant d e n s i t i e s i n cesium c h l o r i d e as o u t l i n e d by k r a u t et a l ( 7 3 ) .  DNA  Using E. c o l l DNA  Schlld-  as a r e f e r e n c e standard,  the samples were each c e n t r i f u g e d a t 4 4 , 0 0 0 rv/min f o r 2 hrs i n the Beckman Model E a n a l y t i c a l u l t r a c e n t r i f u g e and v i o l e t a b s o r p t i o n photographs  taken.  I d e n t i f i c a t i o n of c e l l w a l l sugars. (22)  ultra-  Cummins and  Harris  have r e p o r t e d t h a t i n g e n e r a l , each b a c t e r i a l genus  appears ponents.  to have a c h a r a c t e r i s t i c p a t t e r n of c e l l - w a l l comWith r e g a r d to sugars, f o r example, a l l the  11 C o r y n e b a c t e r i a t e s t e d by these workers, excepting C. pyogenes and  C. haemolyticum, i n c o r p o r a t e d l a r g e q u a n t i t i e s of a r a b i -  nose i n t o the c e l l w a l l .  The l a t t e r two  organisms, i n common  w i t h the genus Streptococcus, l a c k e d arabinose but i n c o r p o r a t e d rhamnose, a sugar which i s not d e t e c t a b l e i n c e l l w a l l p r e p a r a t i o n s of o t h e r  Corynebacterla.  A d e t e r m i n a t i o n of c e l l w a l l sugars was  therefore per-  formed on G. k u t s c h e r l ATCC 1 5 6 7 7 and ATCC 1 5 6 7 8 ,  i n order  t h a t s i m i l a r i t i e s or d i f f e r e n c e s observed might serve to c l a r i f y the r e l a t i o n s h i p between the tvro s t r a i n s . and chromatographic  a n a l y s i s were c a r r i e d out a c c o r d i n g to  the methods d e s c r i b e d i n d e t a i l by Davis and (23).  Hydrolysis  Balrd-Parker  Whole c e l l s were used because, as noted by  these  workers, no o t h e r sugar b e s i d e s r i b o s e i s commonly present i n l a r g e amounts i n the cytoplasm  o r nucleus, and the  r l b o s e sugars d e t e c t e d i n whole c e l l h y d r o l y s a t e s may f o r e be assumed to be c e l l w a l l components. i n c l u d e d i n each r u n to f a c i l i t a t e Examination toxic properties.  Standards  nontherewere  identification.  o f C. k u t s c h e r i c u l t u r e supernatant  for  In view of the v a s t numbers of C. k u t s c h e r l  which are present i n mice when a c t i v e c o r y n e b a c t e r i a l pseudot u b e r c u l o s i s i s i n an advanced stage  ( F i g . 8 ) and the r e l a t i v e l y  m i l d symptoms which accompany the d i s e a s e i t would seem h i g h l y u n l i k e l y t h a t s i g n i f i c a n t l e v e l s of t o x i n c o u l d be produced by the organism.  When death f i n a l l y occurs, gross i n s p e c t i o n  u s u a l l y r e v e a l s o n l y t h a t the s i z e of the abscess or  abscesses  12 has  impaired the a c t i v i t y of one  as the l i v e r ,  o r more v i t a l organs, such  lungs, kidneys, heart or b r a i n .  however, r e p o r t e d an e x o t o x i n so powerful t h a t or i n t r a p e r i t o n e a l i n j e c t i o n of 0.1 c u l t u r e f i l t r a t e was  Wolff  (80),  subcutaneous  ml of a 10"" ^ d i l u t i o n of  s u f f i c i e n t to k i l l  an 18  gm mouse,  d i f f u s e haemorrhages b e i n g found on post-morten examination. Pierce-Chase et a l ( 6 2 ) ,  however, i n j e c t e d 0.1  d i l u t i o n s of u n f i l t e r e d C. k u t s c h e r l not  report  any  ml of  10"  b r o t h c u l t u r e s and  1  did  o b s e r v a t i o n s on post-mortem examination that  were not a t t r i b u t e d to d i f f u s e m u l t i p l i c a t i o n of the  injected  organisms. Preliminary  experiments were t h e r e f o r e  c a r r i e d out  to  determine whether high, l e v e l s of e x o t o x i n were produced i n c u l t u r e by any  of the  Each organism was  C. k u t s c h e r l  inoculated  incubated f o r 4 days a t 3 7 C  s t r a i n s used i n t h i s study.  into veal  A f t e r c e n t r i f u g a t i o n f o r 10  a t 25,000 x g, each supernatant was  sterilized  V M i l l i p o r e f i l t e r and  through a 0.45  i n f u s i o n broth, and •  ml.  These mice were i n s p e c t e d  filtration  injected intraperito-  n e a l l y i n t o a d u l t mice, 6 r e c e i v i n g 0.1 0.5  by  ml  and  6  d a i l y f o r any  receiving v i s u a l symp-  toms of i l l h e a l t h which might be a t t r i b u t e d to t o x i n s filtrate,  but  no  such evidence was  was  repeated w i t h s t r a i n W-l,  was  replaced  by  observed.  except t h a t v e a l  the medium of M u e l l e r and  These workers had  The  had  i n the  experiment  i n f u s i o n broth  Miller  (56).  r e p o r t e d p r o d u c t i o n of d i p h t h e r i c  h i g h potency when C. d l p h t h e r l a e  min  t o x i n of  been grown i n t h i s  13 medium.  Evidence of any  the 4 day  e x o t o x i n having  i n c u b a t i o n p e r i o d was  been produced i n "  s t i l l lacking.  I s o l a t i o n procedures f o r C. k u t s c h e r l . who  Those  have attempted to i s o l a t e C. k u t s c h e r i from  authors  apparently  h e a l t h y but l a t e n t l y i n f e c t e d mice have s t a t e d t h a t such i s o l a t i o n s are e i t h e r i m p o s s i b l e (7t  18,  80).  (34)  o r extremely d i f f i c u l t  In view of the low percentage of success, i t  could be argued i n each case t h a t an undetected f a c t o r " had f i r s t All  "predisposing  activated latency.  the r e p o r t e d i s o l a t i o n attempts, however, have  been made from c u l t u r e s of I n t e r n a l organs.  Experiments  were t h e r e f o r e c a r r i e d out i n t h i s study to d e t e c t the p o s s i b l e presence of v i r u l e n t C. k u t s c h e r l i n the t h r o a t or on the s k i n of l a t e n t l y i n f e c t e d mice. s t r a t e d present  I f C. k u t s c h e r l could be demon-  i n these areas i n a l a r g e percentage of  animals examined, " a c t i v a t i o n of l a t e n t i n f e c t i o n " might i n i t i a l l y i n v o l v e m u l t i p l i c a t i o n of the organisms i n the naso-pharynx f o l l o w e d by d i s s e m i n a t i o n v i a the lungs  or  g a s t r o - i n t e s t i n a l t r a c t when p r e d i s p o s i n g f a c t o r s were present.  Throat  swabs, u s i n g s t e r i l e ,  cotton-tipped  p i c k s , were taken from a n a e s t h e t i z e d mice and  streaked  on v e a l i n f u s i o n agar and blood t e l l u r i t e agar, and immersed and  incubated  toothout  then  i n veal i n f u s i o n broth containing  . 0 5 $ potassium t e l l u r i t e  (Barker and Adamson).  S k i n swabs  were a l s o made from the abdominal r e g i o n of the same animals,  14  the  swab being f i r s t moistened w i t h p h y s i o l o g i c a l s a l i n e .  The  swabs were then t r e a t e d as above.  attempts were made from these  One hundred I s o l a t i o n  regions.  When i s o l a t i o n attempts were made from l i v e r , lungs,  kidneys, heart  ordinary  o r b r a i n , organs were removed u s i n g  a s e p t i c procedures and ground i n a t i s s u e  with veal i n f u s i o n broth. and  Pathogenicity  where d e s c r i b e d  grinder  The mixture was incubated a t 370  made t o v e a l i n f u s i o n agar a f t e r 2 and 4  sub-cultures  days.  spleen,  d e t e r m i n a t i o n s were performed as e l s e -  on a l l t y p i c a l c o r y n e b a c t e r i a l  forms I s o l a t e d .  Pathogenic i s o l a t e s were assumed to be C. k u t s c h e r l . Virulence.  Pierce-Chase et a l ( 6 2 ) demonstrated con-  s i d e r a b l e d i f f e r e n c e s i n the s u s c e p t i b i l i t y of v a r i o u s mouse s t r a i n s t o experimental i n f e c t i o n with a s t r a i n o f C. k u t s c h e r l . In a paper by Fauve et a l which f o l l o w e d  ( 3 4 ) , i s o l a t i o n of  an a v i r u l e n t v a r i a n t of the same b a c t e r i a l s t r a i n was-reported. Because d i f f e r e n c e s o r changes i n the v i r u l e n c e of b a c t e r i a l s t r a i n s and d i f f e r e n c e s i n s u s c e p t i b i l i t y o f mice to i n f e c t i o n by any one of them could have a profound bearing establishment of l a t e n c y ,  on the  the comparative v i r u l e n c e of a l l  s t r a i n s of C. k u t s c h e r l used were determined and the r e l a t i v e s u s c e p t i b i l i t y of t h e three mouse s t r a i n s used a l s o The  comparative v i r u l e n c e o f the v a r i o u s  established.  C. k u t s c h e r l  s t r a i n s was determined by i n j e c t i n g known numbers of the organisms i n 0 . 1 ml of a p h y s i o l o g i c a l s a l i n e suspension  15 Into the l a t e r a l t a l l v e i n s of WSW,  CF-^SPF) o r A/J mice,  i n d i c a t e d i n t a b l e s IV, V, and VI.  V i a b l e counts were  as  first  made of organisms i n 2k hour v e a l b r o t h c u l t u r e s and numbers p r e s e n t r e l a t e d t o the o p t i c a l d e n s i t y a t 650 u, u s i n g the S p e c t r o n i c 20. photometry.  Dosages were t h e r e a f t e r determined by  The comparative s u s c e p t i b i l i t y of the t h r e e  mouse s t r a i n s used was In  spectro-  e s t a b l i s h e d i n the same experiment.  subsequent v e r i f i c a t i o n s of the p a t h o g e n i c i t y of 7  presumed C. k u t s c h e r l i s o l a t e d i n o t h e r experiments,  10'  organisms were i n j e c t e d i n t r a v e n o u s l y i n t o each of t h r e e mice'.  T h i s number of v i r u l e n t C. k u t s c h e r i organisms  was  u n i f o r m l y pathogenic. Latency t r a n s f e r attempts.  Fauve et a l (3*0  have  postulated that i n l a t e n t corynebacterial pseudotuberculosis, the  e t l o l o g i c agent i s harbored i n the organs of i n f e c t e d  mice i n a transformed, a v i r u l e n t  state.  Under c o n d i t i o n s  when the host i s subjected to p h y s i o l o g i c a l s t r e s s , i t i s contended t h a t the a v i r u l e n t form transforms to v i r u l e n c e , and can then be i s o l a t e d as pathogenic C. k u t s c h e r l .  This  theory would account f o r the l a c k o f success experienced i n attempted i s o l a t i o n s o f v i r u l e n t C. k u t s c h e r i from l a t e n t l y i n f e c t e d but a p p a r e n t l y h e a l t h y animals. mation from a v i r u l e n c e to v i r u l e n c e was Fauve, nor was If  In v i t r o  transfor-  not r e p o r t e d by  such t r a n s f o r m a t i o n observed i n t h i s study.  such a t r a n s f o r m a t i o n o c c u r s , i t must be r e c o g n i z e d as of  16  profound importance i n t h e understanding o f the l a t e n t i n corynebacterial pseudotuberculosis.  state  A p r e l i m i n a r y study  i n v o l v i n g the attempted t r a n s f e r o f l a t e n c y i n v i r u l e n t C. k u t s c h e r i f r e e organ homogenates from WSW to C. k u t s c h e r l f r e e mice was t h e r e f o r e I n i t i a t e d . k i l l e d w i t h ether, immersed  Twelve WSW mice were  f o r 5 min i n a 5% aqueous  t i o n o f phenol and washed i n 70$ e t h a n o l .  solu-  The p e r i t o n e a l  c a v i t y o f each mouse was opened, and approximately 1 g of l i v e r and the e n t i r e s p l e e n a s e p t i c a l l y removed. were minced, ground b r i e f l y  These organs  i n a l o o s e - f i t t i n g tissue grinder  and suspended i n 5 nil o f Hanks' balanced s a l t  solution.  Bac-  t e r i o l o g i c a l examinations were made on each p r e p a r a t i o n . 0 . 5 ml o f each homogenate was I n j e c t e d i n t o the peritoneum of each of t h r e e A / J (C. k u t s c h e r l f r e e ) mice, 36 r e c i p i e n t mice i n a l l .  Twelve A / J c o n t r o l s r e c e i v e d s i m i l a r l y prepared  homogenates from A / J mice.  The i n j e c t e d mice r e c e i v e d hydro-  c o r t i s o n e a c e t a t e (Merck Sharp & Dohme) b e f o r e and/or a f t e r the homogenate, w i t h t h e e x c e p t i o n o f animals s e t a s i d e as Cortisone controls.  A t 21 days, a l l mice were s a c r i f i c e d  and examined f o r evidence o f C. k u t s c h e r l i n f e c t i o n .  Attempts  were made t o i s o l a t e C. k u t s c h e r l from the organs of these mice by methods d e s c r i b e d above.  A t the c o n c l u s i o n of t h i s  s e r i e s , the experiment was repeated u s i n g CF^(SPF) mice as r e c i p i e n t s i n p l a c e o f the A / J mice. mice were used as c o n t r o l s .  Likewise, 12  CF^(SPF)  A l l o t h e r c o n d i t i o n s o f the  experiment remained the same.  17 Cortisone  administration.  There a r e s e v e r a l  of a c t i v a t i o n of l a t e n t c o r y n e b a c t e r i a l  reports  pseudotuberculosis  i n mice or r a t s which have been t r e a t e d w i t h c o r t i s o n e 34,  49).  Fauve et a l (3^) found t h a t h y d r o c o r t i s o n e  was much s u p e r i o r i n t h i s r e s p e c t  to s t a r v a t i o n ,  (3,  acetate  pantothenic  a c i d d e f i c i e n c y , water d e p r i v a t i o n , maintenance a t 4C, a d r e n a l i n or endotoxin a d m i n i s t r a t i o n ,  thorotrast  injection,  splenectomy, p a r t i a l hepatectomy, u n i l a t e r i a l l i g a t u r e of the r e n a l v e i n o r X - i r r a d i a t i o n , the l a s t being of a l t e r n a t i v e methods.  Cortisone  the most e f f e c t i v e  i n j e c t i o n s were t h e r e f o r e  used i n t h i s study as a means of v e r i f y i n g l a t e n t coryneb a c t e r i a l pseudotuberculosis mice and conversely,  i n r e p r e s e n t a t i v e groups o f WSW  determining t h a t imported s t r a i n s of  mice ( s t r a i n s CF-^(SPF) and A/J) were f r e e of C. k u t s c h e r i o r g a n i sms. Hydrocortisone acetate  as a s t e r i l e suspension (Merck  Sharp & Dohme) was i n j e c t e d i n t r a p e r i t o n e a l l y , r e c e i v e d a s i n g l e dose of 1 2 . 5  mg,  Some groups  w h i l e o t h e r s were g i v e n  not more than 5 i n j e c t i o n s of 2 . 5 mg each a t 4 day i n t e r v a l s . Mice which s u r v i v e d 21 days were s a c r i f i c e d and examined. Organs were c u l t u r e d f o r i s o l a t i o n of C. k u t s c h e r l as previously  described.  Immunization of mice.  None of the p u b l i c a t i o n s con-  cerned s p e c i f i c a l l y w i t h c o r y n e b a c t e r i a l  pseudotuberculosis  i n mice which have been r e f e r r e d to i n t h i s study ( 3 .  ?> l i t  18  18,  3^. ^3, 47,  48,  49,  62,  63, 80)  have mentioned attempts  to immunize mice against C. kutscherl.  The only reported  immunological experiment with the organism was performed by Fauve et a l (34),  who noted a high t i t e r of C. kutscherl  a g g l u t i n i n produced by immunized rabbits.  Mouse immunization  experiments were carried out i n t h i s study f o r three purposes. 1. To determine whether i n j e c t i o n of k i l l e d C. kutscherl vaccine would confer a s i g n i f i c a n t degree of protection on WSW mice.  This  would be an i n d i c a t i o n of the r o l e of c i r c u l a t i n g antibody, or the lack of i t , i n conditions of resistance to i n f e c t i o n or a c t i v a t i o n of latency respectively. 2. To determine whether a difference existed i n the f i n a l antibody t i t e r of C. kutscheri free mice and l a t e n t l y infected mice when representative groups of these animals were immunized i n i d e n t i c a l programmes.  Is i t  possible, f o r example, that strains of mice which are poor C. kutscherl antibody producers tend to become l a t e n t l y infected? 3. To determine whether the antibody response by mice to C. kutscherl vaccine was s i g n i f i c a n t l y depressed when an immunization programme was i n i t i a t e d i n the erythroblastic stage of Rauscher disease.  Such  19 Immune-depression c o u l d be an Important f a c t o r i n a c t i v a t i o n of corynebacterial p s e u d o t u b e r c u l o s i s d u r i n g t h i s stage of RLV  infection.  B a c t e r i a l v a c c i n e was prepared by standard methods ( 4 4 ) , u s i n g s t r a i n W-l.  B r i e f l y , v e a l i n f u s i o n b r o t h agar  was seeded w i t h the organism, and incubated 48 h r s a t 3 7 C The  c e l l s were washed o f f w i t h normal s a l i n e , washed twice  w i t h normal s a l i n e , heated i n Hanks' balanced cells/ml.  salt  t o 56C f o r JO min and suspended  s o l u t i o n to a d e n s i t y of 5 x 10  The v a c c i n e was t e s t e d f o r s t e r i l i t y by spreading  0.1 ml o f the p r e p a r a t i o n onto a blood agar p l a t e which was then incubated f o r 48 hrs.  M e r t h l o l a t e was then added to a  c o n c e n t r a t i o n of 1:10,000.  Mice were I n j e c t e d i n t r a p e r i -  t o n e a l l y w i t h 0.2 m i s / i n j e c t i o n .  I n j e c t i o n s were normally  a d m i n i s t e r e d twice weekly f o r a three week p e r i o d , f o r a t o t a l of s i x injections.  I n the immunodepression e x p e r i -  ment Rauscher I n f e c t e d mice and c o n t r o l s r e c e i v e d o n l y two injections,  7 and 11 days f o l l o w i n g RLV i n f e c t i o n .  Macrophage s t u d i e s .  I t was r e p o r t e d by Fauve  t h a t mouse p e r i t o n e a l macrophages maintained  (31)  in. v i t r o  a c t i v e l y phagocytosed C. k u t s c h e r i a t a r a t e which was i n d e pendent of whether macrophage were o b t a i n e d from e i t h e r C. k u t s c h e r i f r e e o r l a t e n t l y i n f e c t e d mice. ment was designed  This experi-  t o show whether the rate' o f phagocytosis  20 would vary i f the  source of the macrophages was  the  same  and  pooled sera from d i f f e r e n t groups of mice were added t o  the  c u l t u r e medium.  was  the  source of the macrophage.  normal WSW WSW  CF^(SPF), a G. k u t s c h e r l  Sera were o b t a i n e d from  mice, from C. k u t s c h e r i  mice which had  free strain,  free  CF^ mice, and  been i n f e c t e d with RLV  21 days  from  previously  ( i n f e c t i o n was  v e r i f i e d by r e t i c u l o c y t e count before the  sera  were p o o l e d ) .  A l l mice were 8 weeks o l d .  of  phagocytosis i n cultures be taken as an  supplemented w i t h WSW  i n d i c a t i o n t h a t WSW  which c o n t r i b u t e d  to the r e s i s t a n c e  latently infected strain.  of these mice to i n -  a c h a r a c t e r i s t i c of t h i s  obtained from RLV  mice would i n d i c a t e that t h i s f a c t o r had  may  be a c t i v a t e d  e a r l y weeks by  a f i n d i n g would be an Important one  of  infected  WSW  been l o s t e a r l y  Since l a t e n t c o r y n e b a c t e r i a l  i n the  could  Conversely, a reduced r a t e  p h a g o c y t o s i s where.serum was  infection.  serum  serum contained a f a c t o r  j e c t i o n of v i r u l e n t C. k u t s c h e r l ,  the RLV  A higher r a t e  RLV  in  infection  i n f e c t i o n , such  i n the c o n s i d e r a t i o n  of  f a c t o r s Inducing a c t i v e i n f e c t i o n . Mice were k i l l e d w i t h d i e t h y l ether and o b t a i n e d a s e p t i c a l l y from the p e r i t o n e a l w i t h Hanks  1  centrifuging  c a v i t y by washing  balanced s a l t s o l u t i o n c o n t a i n i n g  sodium h e p a r i n (Upjohn)/ml.  The  5 minutes at low  minimal medium ( D i f c o )  10 u n i t s  of  c e l l s were sedimented  by  speed, resuspended i n Eagle's  containing  w i t h a c o n c e n t r a t i o n of 10  c e l l s were  10$  cells/ml.  f e t a l c a l f serum (Hyland) The  cell  suspension  21 was  p i p e t t e d i n t o s t e r i l e L e l g h t o n tubes c o n t a i n i n g cover-  slips.  The tubes were t i g h t l y s e a l e d with rubber stoppers,  and incubated a t 3 7 C became a c i d i c .  The medium was  changed whenever i t  A f t e r 48 hrs those c e l l s which adhered  the g l a s s were assumed to be v i a b l e macrophage ( 3 8 ) . k i l l e d b a c t e r i a were added to a f i n a l 2 x io-7/nl and i n c u b a t i o n was minutes. i n Hanks  Phagocytosis was 1  balanced s a l t  were immediately  to Heat  c o n c e n t r a t i o n of  continued f o r from 1 to 60  stopped by r i n s i n g the  coverslips  s o l u t i o n and. then the a d h e r i n g  fixed i n  ether:95% ethanol 1:1.  The  cells cover-  s l i p s were then Giemsa s t a i n e d , washed i n running water and mounted on s l i d e s . standard methods. Serology.  P h a g o c y t i c index was determined  by  (See Appendix I I I ) . The need f o r immunological  d a t a i n any  c o n s i d e r a t i o n of the f a c t o r s c o n t r i b u t i n g to maintenance o r a c t i v a t i o n of l a t e n t I n f e c t i o n has been d i s c u s s e d under Immunization  procedures.  f i c u l t y encountered was  I n t h i s connection, a major d i f to d e v i s e a s e n s i t i v e and r e p r o -  ducible s e r o l o g i c a l t e s t f o r determining c i r c u l a t i n g  anti-  body t i t e r . B a c t e r i a l a g g l u t i n a t i o n , i n g e n e r a l b e i n g the e a s i e s t to perform, was was  the f i r s t  found d i f f i c u l t  technique attempted.  However, i t  to get C. k u t s c h e r l organisms  i n the t e s t i n t o a s t a b l e suspension.  Partial  f o r use  auto-agglu-  t i n a t i o n i n v a r i a b l y o c c u r r e d d u r i n g the course of the  22 reaction.  Complement f i x a t i o n and p a s s i v e hemagglutination  were then attempted,  and of the two, p a s s i v e hemagglutination  was found to be the most r e p r o d u c i b l e .  Immunofluorescent  s t a i n i n g was used t o v e r i f y the r e s u l t s obtained i n p a s s i v e h e m a g g l u t i n a t i o n i n a q u a l i t a t i v e way, and a l s o , immunological strains  to,observe  c r o s s - r e a c t i o n of the d i f f e r e n t C. k u t s c h e r i  used.  Sheep e r y t h r o c y t e s (BBL) were prepared f o r p a s s i v e h e m a g g l u t i n a t i o n by standard methods ( 1 7 ) .  The t a n n i c a c i d  t r e a t e d e r y t h r o c y t e s were coated w i t h C. k u t s c h e r i a n t i g e n Q  prepared by s o n i c a t i o n of 10 ml of a l O V m l s a l i n e p e n s i o n o f washed c e l l s c h i l l e d treatment.  sus-  i n an i c e water bath d u r i n g  C. k u t s c h e r l a r e r e s i s t a n t t o s o n i c a t i o n and  treatment was continued f o r 15 i n t e r v a l s of 1 minute a t an i n t e n s i t y o f 70 on the B r o n w i l l B i o s o n i c . was c e n t r i f u g e d f o r 5 minutes a t 2 , 5 0 0  The suspension  x g to remove most  whole c e l l s and clumps and the supernatant d i l u t e d and titrated  t o o b t a i n the optimum c o n c e n t r a t i o n of a n t i g e n f o r  c o a t i n g tanned c e l l s , u s i n g the method of S t a v l t s k y Immunofluorescent s t a i n i n g was performed  (77).  on h e a t - f i x e d  smears of washed b a c t e r i a l c e l l s on cover s l i p s .  These were  covered w i t h mouse antiserum t o C. k u t s c h e r l f o r 10 min, washed i n s a l i n e f o r 1 min, covered w i t h f l u o r e s c e l n conjugated goat antiserum to mouse  ^globulin  (Hyland) f o r  an a d d i t i o n a l 10 min, and a g a i n washed i n s a l i n e f o r 1 min. The  c o v e r s l l p s were mounted on g l a s s s l i d e s w i t h Bacto FA  23 mounting f l u i d and  observed and photographed through a  Z e i s s u l t r a v i o l e t microscope. Clearance s t u d i e s .  ,  Observations on the clearance  of  b a c t e r i a from the blood of v a r i o u s animals which have been injected intravenously  have shown that b a c t e r i a can  c l e a r e d v e r y r a p i d l y and  i n l a r g e numbers by the  be  reticulo-  e n d o t h e l i a l system (RES), t h a t p r e v i o u s immunization g r e a t l y improves the a b i l i t y of the RES organisms, and clearance  to remove and  kill  ingested  that i n the case of v i r u l e n t organisms, r a p i d  i s followed  by a second phase when the blood con-  c e n t r a t i o n of b a c t e r i a i n c r e a s e s  (4).  These s t u d i e s were  undertaken to determine whether there might be s i g n i f i c a n t d i f f e r e n c e s i n the r a t e s at which v i r u l e n t C. k u t s c h e r l c l e a r e d by the RES f r e e mice, and  of l a t e n t l y i n f e c t e d mice, C.  are  kutscheri  mice i n the e a r l y stages o f Rauscher  disease.  I f such d i f f e r e n c e s were noted, they might be u s e f u l i n e x p l a i n i n g the Increased r e s i s t a n c e o f l a t e n t l y i n f e c t e d mice and  the decreased r e s i s t a n c e of RLV  C. k u t s c h e r i  s t r a i n 1 5 6 7 7 was  i n f e c t e d mice.  l a b e l e d w i t h P-  32  (Radio-  chemical Center, Amisham) by growing the c e l l s i n v e a l i n f u s i o n broth  containing  1 mc  p 3 / 2 0 0 ml medium. 2  After  48 h r s i n c u b a t i o n a t 3 7 C the l a b e l e d c e l l s were prepared f o r i n j e c t i o n as d e s c r i b e d Q imately  2.5  x 10  by Benacerraf et a l ( 4 ) .  Approx-  c e l l s were Introduced i n t o each mouse by  Injection into a l a t e r a l t a i l vein.  At i n t e r v a l s t h e r e a f t e r ,  24 0 . 0 5 ml samples of b l o o d were taken from the i n j e c t e d mouse by r e t r o o r b i t a l puncture a c c o r d i n g to the technique d e s c r i b e d by R i l e y planchet.  (67).  Each sample was  spread onto a separate  A l l p l a n c h e t s were saved u n t i l the c o n c l u s i o n of  the experiment and the r a d i o - a c t i v e count on each determined w i t h a P h i l l i p s PW  4032 Counter u s i n g a Geiger tube.  Mice used i n c l e a r a n c e s t u d i e s i n c l u d e d of the WSW  and the A / J s t r a i n s ; and WSW  "normal" mice  mice which  r e c e i v e d RLV a t the neonatal stage o r as weanlings.  had Rates  of c l e a r a n c e were compared. E l e c t r o n microscopy.  Histological preparations  observed by l i g h t microscopy r e a d i l y showed the presence of C. k u t s c h e r l i n i n f e c t e d mouse t i s s u e  (Pig. 10,  p a r t i c u l a r i n t e r e s t i n t h i s study, however, was of the microorganism w i t h i n phagocytosing c e l l s ,  11,  12).  Of  the s t a t e i . e . , were  the c l u s t e r s of i n g e s t e d b a c t e r i a d i s i n t e g r a t i n g or were they predominantly i n t a c t and p o s s i b l y m u l t i p l y i n g ?  Fauve  has a l r e a d y shown t h a t C. k u t s c h e r i can m u l t i p l y w i t h i n p e r i t o n e a l macrophages  (32).  When C. k u t s c h e r l i s i n j e c t e d i n t r a v e n o u s l y , m u l t i p l e abscesses u s u a l l y appear i n the r e n a l c o r t e x ( F i g . 1 1 ) , Depending  on the numbers of organisms  i n j e c t e d and the r e -  s i s t a n c e of the mouse s t r a i n used, these abscesses sometimes r e g r e s s ( 6 2 ) , f e c t e d kidney was  I t was  may  f o r t h i s reason that i n -  s e l e c t e d as t i s s u e i n which to study  p h a g o c y t o s i s by c e l l s o t h e r than p e r i t o n e a l macrophage.  25 E l e c t r o n microscopy was used because e l e c t r o n micrographs more r e a d i l y show whether i n g e s t e d  b a c t e r i a l c e l l s are  i n t a c t o r undergoing c y t o l y s i s ( F i g . 18). Mice were k i l l e d w i t h ether.  Kidneys were immediately  removed, d i c e d and f i x e d f o r 20 min i n a 4$ s o l u t i o n of g l u t a r a l d e h y d e i n 0.0?M phosphate b u f f e r a t pH 7.2. preparation  The  was washed i n b u f f e r and p o s t - f i x e d 45 min i n  2% Osmium t e t r o x i d e  (45).  B a c t e r i a l c o l o n i e s were f i x e d  i n s i t u f o r 5 min w i t h 4$ glutaraldehyde, i n agar, t r a n s f e r r e d t o K e l l e n b e r g e r 16 h r s . A f t e r f i x a t i o n , p r e p a r a t i o n s  cut out, enrobed  f i x a t i v e and f i x e d f o r were washed i n more  phosphate b u f f e r , dehydrated w i t h a graded s e r i e s o f a l c o hols,  soaked i n 3 changes of propylene oxide and embedded  i n epon 812 by the method of L u f t  (50).  T h i n s e c t i o n s were  cut w i t h g l a s s k n i v e s on a Porter-Blum MT-2  ultramicrotome,  p i c k e d up on carboned g r i d s and s t a i n e d w i t h u r a n y l follovred by l e a d c i t r a t e .  acetate  The g r i d s were examined w i t h a  P h i l l i p s EM 100C E l e c t r o n microscope.  26 Results Virus t i t r a t i o n .  Using the Reed and Muench method  f o r d e t e r m i n i n g 50% endpoint WSW  '  ( 6 6 ) , the ID^Q f o r neonatal  mice was c a l c u l a t e d to be 1 0 ~ 2 . 2 p  e r  o . l ml.  The r e s u l t s  of t h i s experiment are presented i n d e t a i l i n Table I (the second to the f i f t h group of mice i n c l u s i v e ) and the d e t a i l s of the c a l c u l a t i o n are s e t f o r t h i n Appendix I .  The  t i o n r e s u l t s are interpreted g r a p h i c a l l y i n F i g .  1.  The WSW  titra-  s t r a i n of mice were found to be c o n s i d e r a b l y  more r e s i s t a n t to RLV than the BALB/c mice from which t h i s v i r u s was harvested and w i t h which s t r a i n the v i r u s had o r i g i n a l l y been t i t r a t e d .  The r e p o r t e d i n f e c t i v i t y  f o r weanling BALB/c mice was 1 0 ~ 5 . 5 p  e r  l . o ml.  A  titer compar-  a b l e f i g u r e f o r WSW  neonatal mice estimated by v i s u a l i n -  s p e c t i o n o f F i g . 1,  would be approximately 10""3.  j  n  view  of o t h e r r e p o r t s which have been made on random-bred mice (27),  t h i s d i f f e r e n c e i s not s u r p r i s i n g . Because WSW  mice were most e x t e n s i v e l y used i n the  experiments o f t h i s study which i n v o l v e d RLV i n j e c t i o n , a l l 2 9 dosages r e p o r t e d are based on the t i t e r of 10 mined w i t h these mice.  *^ d e t e r -  No m i c r o b i a l growth of any k i n d  r e s u l t e d from p l a t i n g out and i n c u b a t i n g a p o r t i o n o f the virus.  Blood agar p l a t e s were incubated a e r o b i c a l l y and  a n a e r o b i c a l l y a t 37C f o r 72 h r s .  <9  i -I  i  -2 LOG  I  i  L  -3  -4  -5  DILUTION  P i g . 1. Rauscher leukemia v i r u s I D ^ Q d e t e r m i n a t i o n . A t o t a l o f 117 neonatal mice were i n j e c t e d i n t h i s e x p e r i ment (groups 2 to 5 i n c l u s i v e , Table I ) , each group r e c e i v i n g 0 . 1 ml of one of the d i l u t i o n s i n d i c a t e d . The curve f o l l o w s the average s p l e e n weight f o r each group 21 days folloxving v i r u s a d m i n i s t r a t i o n . The dashed h o r i z o n t a l l i n e i s a t 300 mg and r e p r e s e n t s the c r i t e r i o n f o r splenomegaly. The dashed v e r t i c a l l i n e I n d i c a t e s the c a l c u l a t e d t i t e r of the v i r u s u s i n g the Reed and Muench method f o r e s t i m a t i n g 50$ endpoint.  28 TABLE I Rauscher Leukemia V i r u s A d m i n i s t r a t i o n and the Incidence of C o r y n e b a c t e r i a l P s e u d o t u b e r c u l o s i s i n , and the I s o l a t i o n of .C. k u t s c h e r i from, v a r i o u s s t r a i n s of mice.  * Mouse No. of S t r a i n Mice Sex Age WSW  #* Log Dose (ID ) 5 Q  *** Splenomegaly  # Pseudotuberculosis  12  N  2.0  12  0  27  N  1.0  24  1  26  N  0  12  1  27  N  -1.0  3  0  30  N  -2.0  3  0  7  N  -3.0  0  0  ## Evidence of C.k. i n organs 1/12  (continued)  * Age. (At the time RLV was a d m i n i s t e r e d ) . 1, Neonatal; W, Weanling ( 4 - 5 weeks o l d ) ; and A, Adult ( 8 - 1 2 weeks o l d and over 25 g w e i g h t ) . ** RLV Dose ( I D t n ) . Expressed as a m u l t i p l e of the q u a n t i t y r e q u i r e d t o i n f e c t 50$ of neonatal WSW mice, w i t h splenomegaly a t 21 days a f t e r i n j e c t i o n , the c r i t e r i o n of i n f e c t i o n . The v i r u s was suspended i n Hanks' s o l u t i o n and i n j e c t e d subcutaneously i n t o neon a t a l mice and i n t r a p e r i t o n e a l l y i n t o weanling o r a d u l t mice. *** Splenomegaly. Spleen weight at time of s a c r i f i c e over 300 mg. Mice i n j e c t e d as n e o n a t a l s were s a c r i f i c e d a t 21 days, o r , i f i n j e c t e d as weanlings o r a d u l t s , a t 28 days. # P s e u d o t u b e r c u l o s i s . A c t i v e c o r y n e b a c t e r i a l pseudot u b e r c u l o s i s , w i t h r u f f l e d f u r , waddling g a i t , and presence of caseous abscesses i n o r a s s o c i a t e d w i t h l i v e r , lungs o r o t h e r organs. ## Evidence of C. k u t s c h e r l i n organs. An estimate based on m i c r o s c o p i c o b s e r v a t i o n of c o r y n e b a c t e r i a l forms i n organs, j u s t i f i e d by C. k u t s c h e r i c u l t u r e s made from a r e p r e s e n t a t i v e number (see accompanying d i s c u s s i o n ) .  29 TABLE I (continued)  Mouse No. of S t r a i n Mice Sex WSW  18 2k  17  0  2/18  w  1.7  20  1  3/12  Pseudotuberculosis  ## Evidence of C k . i n organs t  w  2.0  18  1  3/18  5k  cf  w  1.7  39  2  5/36  9  A  2.0  5  0  2/12  9  A  1.7  k  0  1/12  9  A  -  0  0  0/18  12  cf  A  2.0  3  1  1/12  36  cf  A  1.7  9  1  3/36  18  cf  A  -  0  0  0/18  12  cf  A  1.7  3  0  0/12  20  cf  A  1.7  11  0  0/20  18  A/J  2.0  #  cf  12  1  w  5 0  #** Splenomegaly  18  12  CF  9  * Age  *# Log Dose (ID )  P l e a s e see the p r e v i o u s page f o r an e x p l a n a t i o n of the column headings.  30  ^ ^ ^ ^ ^ ^ ^ ^ ^ ^  F i g . 2. L i v e r and s p l e e n of a WSW mouse which has developed c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s f o l l o w i n g RLV infection. C. k u t s c h e r l a r e present i n numerous t u b e r c l e l i k e abscesses d i s t r i b u t e d i n t h e l i v e r . The extremely distended s p l e e n i s t y p i c a l o f the l a t e prelymphoid phase of the Rauscher d i s e a s e .  F i g . 3. Spleens removed from RLV i n f e c t e d mouse and a non-infected c o n t r o l 28 days p o s t - i n f e c t i o n . Splenomegaly i s the g e n e r a l l y accepted c r i t e r i o n f o r e s t a b l i s h i n g Rauscher disease. The r e s p e c t i v e weights a r e 2,606 and 206 mg.  31 Rauscher leukemia v i r u s Induced c o r y n e b a c t e r i a l pseudotuberculosis. t u b e r c u l o s i s was  RLV  f i r s t observed  gross i n s p e c t i o n of 117 i n j e c t e d w i t h RLV  as neonatals  f r e q u e n t l y observed  c o r y n e b a c t e r i a l pseudoi n our l a b o r a t o r y d u r i n g  21-day o l d WSW  stock of Rauscher v i r u s .  Rauscher d i s e a s e  induced  mice which had been  i n a t i t r a t i o n of a  new  An enlarged and mottled l i v e r i s  d u r i n g the e r y t h r o b l a s t i c stage of the  (65),  but i n two  i n s t a n c e s noted,  there  were areas of coalescence w i t h a g e n e r a l l y n e c r o t i c appearance.  Gram s t a i n s of g r a n u l a r pus removed from the l e s i o n s  r e v e a l e d an abundance of gram p o s i t i v e b a c i l l i which were subsequently kutscheri  (Fig. 10)  i d e n t i f i e d as Corynebacterium  (Table I I I ) .  I n s t u d i e s of RLV  i n d u c t i o n which f o l l o w e d ,  (Table I ) ,  an a d d i t i o n a l 230 mice were i n j e c t e d w i t h 50 o r 100 the v i r u s .  T h i s number i n c l u d e s 12  mice, none of which were expected pseudotuberculosis were a l s o examined. the study  and  36 WSW  There are t h e r e f o r e 198  WSW  controls mice i n  i n which a c t i v a t i o n o f l a t e n c y could be  approximately  of  to develop c o r y n e b a c t e r i a l  expected.  (with r u f f l e d f u r , waddling  g a i t and l e s i o n s i n one o r more i n t e r n a l organs) i n 6,  0  20 A/J  CF-^SPF) mice and  none o f which d i d .  Of t h i s number, o v e r t d i s e a s e  ID^  developed  3$.  In a d d i t i o n to gross i n s p e c t i o n , Giemsa or Gram s t a i n e d touch p r e p a r a t i o n s were made from the l i v e r , lungs and kidneys of 158  of the 198  mice.  Of t h i s  spleen, 158,  »  32 c o r y n e b a c t e r i a l forms were d e f i n i t e l y i d e n t i f i e d i n 2 1 .  In  one experiment,  v e a l i n f u s i o n b r o t h c u l t u r e s were a l s o made  from the l i v e r ,  spleen, lungs and kidneys of 36 RLV i n f e c t e d  mice ( i n j e c t e d as weanlings and s a c r i f i c e d 28 days a f t e r infection).  G. k u t s c h e r l grew I n a l l o f 5 cases where  c o r y n e b a c t e r i a l forms were i d e n t i f i e d i n h i s t o l o g i c a l p r e p a r a t i o n , and i n 2 cases where no c o r y n e b a c t e r i a l forms were seen.  I t should t h e r e f o r e be s a f e to assume that C. k u t s c h e r i  was p r e s e n t i n the organs o f a t l e a s t 21 o f 158 mice examined, o r approximately 1 3 $ . The above percentages a r e based on a l l i n j e c t e d mice examined, r e g a r d l e s s of whether was demonstrable  a t the time o f s a c r i f i c e .  mice which developed a c t i v e c o r y n e b a c t e r i a l  the RLV splenomegaly  However, a l l pseudotuber-  c u l o s i s had spleens weighing over 5 0 0 mg and a h i g h p e r c e n t age o f n u c l e a t e d c e l l s i n t h e p e r i p h e r a l blood.  Therefore,  the percentage o f mice w i t h confirmed Rauscher d i s e a s e which developed a c t i v e p s e u d o t u b e r c u l o s i s , i n c l u d i n g t h e animals i n the o r i g i n a l v i r u s t i t r a t i o n experiment, was 8 out of 163,  which i s approximately 5 $ .  & comparable f i g u r e f o r  estimated presence of v i r u l e n t C. k u t s c h e r l i n the i n t e r n a l organs of Rauscher  i n f e c t e d mice would be 21 out of 1 2 7 o r  16.5$. In a l l occurrences of a c t i v e p s e u d o t u b e r c u l o s i s claimed, i d e n t i f i c a t i o n of the c a u s a t i v e agent was confirmed by b i o c h e m i c a l t e s t s , and a l l the i s o l a t e s were a l s o  33  confirmed as pathogenic, producing nodular abscesses i n the r e n a l c o r t e x on i n t r a v e n o u s i n j e c t i o n .  With regard to the  d i s t r i b u t i o n of the advanced l e s i o n s , of the 8 cases confirmed, 3 i n v o l v e d the l i v e r and  s p l e e n and two  the l i v e r  alone, 2 i n v o l v e d one of the lungs and one l a r g e abscess was  l o c a t e d i n the r i g h t hemisphere o f the b r a i n .  l a t t e r mouse c i r c l e d c o u n t e r - c l o c k w i s e and was p a r a l y s e d on the l e f t  The  assumed  side.  These data are c o n s i d e r e d as c o n v i n c i n g evidence t h a t RLV  i n f e c t i o n can induce or a c t i v a t e  corynebacterial  p s e u d o t u b e r c u l o s i s i n a s t r a i n of l a t e n t l y i n f e c t e d mice. F u r t h e r , because m u l t i p l i c a t i o n of C. k u t s c h e r i w i t h i n the organs of mice which were examined i s e v i d e n t 3 - 4 weeks after infection,  i t i s a l s o c l e a r that i n d u c t i o n i s t a k i n g  p l a c e d u r i n g the e r y t h r o b l a s t i c o r e a r l y stage of the Rauscher disease.  Although the approximate  age and the sex of the  mice used i s i n d i c a t e d i n Table I as a matter of course, f u r t h e r breakdown of the data would r e q u i r e the use of a great many more mice i f the r e s u l t s were to be significant.  Since both sexes and a l l ages were i n v o l v e d ,  t h i s aspect was  not pursued  R e t i c u l o c y t e counts. experiment  was  further. The  i n i t i a l purpose of t h i s  to i n v e s t i g a t e r e t i c u l o c y t o s i s as a method  f o r d e t e r m i n i n g Rauscher d i s e a s e i n RLV i n s t u d i e s of immunodepression. male WSW  statistically  i n j e c t e d mice used  A group of 12  mice which were i n j e c t e d w i t h 50 I D ^  0  5-week o l d of  RLV  3^ (Table I I ) , had an average r e t i c u l o c y t e count of 1.7% a t the b e g i n n i n g o f the experiment, 4 . 9 $ a t 1 4 days, 5.2% a t 2 8 days, and 9 . 8 $ a t 4 2 days.  The range a t 1 4 days was  from 1 . 6 to 13.0. and counts a l s o v a r i e d over a wide range i n the same mouse from week to week. I t i s important to c o n s i d e r t h a t WSW  i s a random bred  stock of mice which l a c k s u n i f o r m i t y even on v i s u a l  inspec-  t i o n , and could thus be expected to show g r e a t e r I n d i v i d u a l v a r i a t i o n s of response to I n f e c t i o n than an inbred l i n e .  In  F i g . 4 . R e t i c u l o c y t e s i n the p e r i p h e r a l blood of RLV i n f e c t e d mice 1 4 days f o l l o w i n g i n t r a p e r i t o n e a l i n j e c t i o n o f 50 TD50 of v i r u s i n t o a d u l t WSW mice. R e t i c u l o c y t e s a r e immature e r y t h r o c y t e s c o n t a i n i n g n u c l e a r remnants, and the presence of l a r g e numbers i n p e r i p h e r a l blood i s an i n d i c a t i o n of e x c e s s i v e e r y t h r o p o i e t i c a c t i v i t y , such as o c c u r s i n the e r y t h r o b l a s t i c stage of the Rauscher d i s e a s e . B r i l l i a n t c r e s y l blue and Wright's s t a i n , x 1200.  35  TABLE I I Reticulocyte  Counts  Percentage R e t i c u l o c y t e s Observed of T o t a l Blood C e l l s Counted Mouse No.  Day 14  Day 28  Day 42  1  13.0  2.6  12.6  2  3-7  5.0  12.8  3  2.4  2.8  18.5  4  2.7  3.6  13.0  5  1.6  5.2  5.6  6  -  8.6  6.0  7  1.9  3.0  7.8  8  3.8  6.5  4.2  9  12. 3  6.8  6.2  3.6  2.8  5.7  3.2  11.2  12.2  11.8  10 11 12  4.6  R e t i c u l o c y t e counts shown were performed on the p e r i p h e r a l blood of mice which had been i n j e c t e d a t Day 0 w i t h 100 ID50 °f RLV. Blood was s t a i n e d w i t h b r i l l i a n t c r e s y l blue ana c o u n t e r s t a i n e d w i t h Wrights. At l e a s t 1 , 0 0 0 c e l l s were counted f o r every percentage which appears i n the table. The mice were 5 weeks o l d a t the b e g i n n i n g of the experiment, and had an average r e t i c u l o c y t e count o f 1 . 7 $ . The average count i s p l o t t e d i n P i g . 5 .  36  co  15  UJ  I>o O _j 3 O UJ  tr  _l 14 DAYS  POST  —I 28  42  INFECTION  F i g . 5. P r o g r e s s i v e r e t i c u l o c y t o s i s i n the Rauscher disease. 50 ID50 of RLV was a d m i n i s t e r e d to each of 12 WSW mice by i n t r a p e r i t o n e a l i n j e c t i o n . The curve i n d i c a t e s the average percentage of r e t i c u l o c y t e s i n the t o t a l blood c e l l s counted f o r the 12 experimental mice on the days indicated.  37 s p i t e of the l a c k of u n i f o r m i t y ,  r e t l c u l o c y t o s i s was  never-  t h e l e s s concluded to be u s e f u l i n the d e t e r m i n a t i o n o f e a r l y Rauscher i n f e c t i o n .  In the experiments designed to show  immunodepression of C. k u t s c h e r i antibody s y n t h e s i s i n g RLV  i n f e c t i o n , i t was  count of 3 . 4 $  follow-  observed t h a t when a r e t i c u l o c y t e  (twice the normal average) was  used as  c r i t e r i o n , d e f i n i t e immunodepression occurred  a  (Table  IX).  There i s a l s o a c o r r e l a t i o n between r e t l c u l o c y t o s i s and splenomegaly, but more extensive l i n e of mice, would be r e q u i r e d r e l a t i o n s h i p was  s t u d i e s , u s i n g an  to determine whether t h i s  a quantitative  Microorganisms and  one.  cultivation.  C. k u t s c h e r l d e s i g n a t e d as Wl and l a t e d from WSW  mice d u r i n g  were the only two  the  inbred  W2  The  s t r a i n s of  were two  of many i s o -  course of t h i s study,  which were examined i n t e n s i v e l y .  but Bio-  chemical r e a c t i o n s and o t h e r p r o p e r t i e s of these two were examined and  compared w i t h the two  strains  imported s t r a i n s of  C. k u t s c h e r l used i n t h i s study, jtfTCC 1 5 6 7 7 and ATCC 1 5 6 7 8 . The  r e s u l t s of a l l these t e s t s are set f o r t h i n Table I I I .  The  l a s t page of Table I I I a l s o d e s c r i b e s  t e s t s were c a r r i e d out. to v e r i f y the  The  t e s t s were performed not  these only  i d e n t i f i c a t i o n of i s o l a t e s made i n t h i s study,  but a l s o f o r the  sake of comparing f u r t h e r ATCC 1 5 6 7 7 ,  i s pathogenic f o r mice) and ATCC 1 5 6 7 8 , variant  b r i e f l y how  (non pathogenic) to see  (which  i t s presumed a v l r u l e n t  i f s i m i l a r i t i e s would be  38  found i n the c e l l w a l l composition would support  with DNA base r a t i o which  the v a r i a n t h y p o t h e s i s .  As i s shown i n  s e c t i o n s 4 and 5 of Table I I I , no evidence  to support  such  a h y p o t h e s i s was found. The major d i f f e r e n c e s observed were as f o l l o w s : S t r a i n Wl showed s t r o n g l y p o s i t i v e n i t r a t e r e d u c t i o n i n 12 hrs,  W2 grew r a p i d l y on a r t i f i c i a l media and was more r e s i s -  t a n t to p e n i c i l l i n than the o t h e r s t r a i n s , ATCC 1 5 6 7 7 was a slow reducer  of n i t r a t e  (48 hrs to a weakly p o s i t i v e r e a c t i o n )  and was more s e n s i t i v e to p e n i c i l l i n . l a s e negative,  ATCC 15678 was c a t a -  reduced the l i t m u s dye i n l i t m u s milk, and  was a c h a i n i n g c o c c o i d organism, q u i t e u n l i k e the o t h e r strains. The  f i r s t three were s i m i l a r i n p a t h o g e n i c i t y f o r  mice; ATCC 15678 was non pathogenic strains testd.  Pathogenicity  f o r any o f the mouse  i s d i s c u s s e d f u r t h e r i n the  following section. The  l a t t e r s t r a i n was a l s o u n l i k e  "conventional"  C. k u t s c h e r l i n t h a t g a l a c t o s e and rhamnose, and not g a l a c t o s e and arabinose,  were the major c e l l w a l l sugars  (Table I I I and F i g . 7 ) .  I n t h i s r e s p e c t the v a r i a n t r e -  sembles C. pyogenes and C. haemolytlcum.  Furthermore, ATCC  1 5 6 7 7 has a n u c l e i c a c i d base r a t i o which d i f f e r s from that r e p o r t e d f o r C; k u t s c h e r i ( 1 2 ) . base r a t i o  (GC) of ATCC 1 5 6 7 7 ,  as estimated  considerably  Thus, the DNA from buoyant  d e n s i t y i n cesium c h l o r i d e , i s 5 8 . 2 , and t h a t o f ATCC 15678  39  TABLE I I I Biochemical Reactions and other p r o p e r t i e s of v a r i o u s C. k u t s c h e r i s t r a i n s Test o r P r o p e r t y Analysed (1)  S t r a i n o f Corynebacterlum k u t s c h e r l Wl W2 ATCC15677 ATCCI5678  Fermentations Adonitol  +  +  +  +  Arabitol  +  +  +  +  Arabinose  +  +  +  +  Dextrin  +  +  +  +  Dextrose  +  +  +  +  Glycerol  -  -  -  +  +  Inulin Lactose  ±  ±  +  +  Maltose  +  +  +  +  Manitol  +  +  +  Rafflnose  ±  ±  ±  +  Salicin  +  +  +  +  Sucrose  +  +  +  +  Xylose  +  +  +  Chloramphenicol  S  S  S  S  Penicillin  S  R  S  S  Streptomycin  S  S  S  MS  Tetracycline  S  S  S  MS  Thiosulfll  S  S  S  R  (2) S e n s i t i v i t y :  40 TABLE I I I colit inued Test o r P r o p e r t y Analysed  S t r a i n of Corynebacterium Wl W2 ATCC 15677  kutscheri; ATCC 15678  (3). Other:  +  +  +  -  - .  -  Hemolysis  -  -  •-  HgS p r o d u c t i o n  -  Indole  -  -  -  -  -  +  +  +  +  +  +  Nutrient broth  gran.  gran.  gran.  Tellurite  +  +  +  Nitrate red.  +  +  +  Catalase Gelatin  liq.  Litmus m i l k 6.5$  NaCl  Nutrient  agar  (4) DNA base r a t i o (GC): (5)  C e l l Wall  -  BR  + stringy +  58.2  42.0  +  +  sugars:  Arab!nose Galactose Rhamnose (6) Pathogenicity f o r mice  N.B.  P l e a s e see f o l l o w i n g page f o r d e s c r i p t i o n and i n t e r p r e t a t i o n of t e s t s .  +  41 D e s c r i p t i o n o f t e s t s o r p r o p e r t i e s g i v e n i n Table I I I . Fermentation r e a c t i o n s were c a r r i e d out by I n o c u l a t i n g b a c t e r i a i n t o tubes o f Bacto p u r p l e b r o t h base, each c o n t a i n i n g 1 . 0 $ o f the fermentable s u b s t r a t e as l i s t e d i n the f i r s t column. The tubes were read a f t e r 7 days i n c u b a t i o n a t 37C and r e p o r t e d as a c i d p r o d u c t i o n +, no change -, d o u b t f u l +. S e n s i t i v i t y t e s t s were performed by seeding a lawn of the t e s t organism on blood agar and s e t t i n g Bactos e n s i t i v i t y d i s c s as f o l l o w s : chloramphenicol 5 meg, p e n i c i l l i n 5 u n i t s , d i h y d r o s t r e p t o m y c i n 2 meg, t e t r a c y c l i n e 5 meg, and t h i o s u l f i l 1 mg. Reactions were r e p o r t e d as f o l l o w s : zone o f i n h i b i t i o n over 1 . 5 cm diameter, s e n s i t i v e ( S ) ; l e s s than 1 . 5 cm = moderately s e n s i t i v e (MS); no i n h i b i t i o n = r e s i s t a n t (R). Other t e s t s were c a r r i e d out as f o l l o w s ( p o s i t i v e r e a c t i o n +, negative r e a c t i o n -, d o u b t f u l +); C a t a l a s e +, p r o d u c t i o n of O 2 when H 2 O 2 added t o 48 hr b r o t h c u l t u r e ; G e l a t i n l i q u l f l c a t i o n +, l i q u i f i c a t i o n of n u t r i e n t g e l a t i n w i t h i n 7 days; Hemolysis +, zones of c l e a r i n g around c o l o n i e s on blood agar; H 2 S + , b l a c k c o l o r a t i o n o f stabbed l e a d a c e t a t e agar w i t h i n 7 days; i n d o l e + , Indole d e t e c t e d i n t r y p t o n e b r o t h a f t e r 7 days; Litmus milk + , a c i d p r o d u c t i o n w i t h i n 7 days, RR = r a p i d r e d u c t i o n ( l i t m u s dye reduced a f t e r o v e r n i g h t i n c u b a t i o n ) ; 6 . 5 $ NaCl +, growth i n 6 . 5 $ NaCl n u t r i e n t b r o t h w i t h i n 7 days; N u t r i e n t agar +, growth w i t h i n 7 days; N u t r i e n t broth, gran, ( g r a n u l a r ) o r s t r i n g y d e s c r i b e the sediment i n b r o t h c u l t u r e ; T e l l u r i t e +, b l a c k c o l o n i e s on 0 . 0 5 $ t e l l u r i t e agar w i t h i n 7 days; N i t r a t e r e d u c t i o n +, d e t e c t i o n o f n i t r i t e i n n u t r i e n t b r o t h c o n t a i n i n g 0 . 1 $ KNO3 a f t e r 7 days. DNA base r a t i o (GC) was determined from the r e s p e c t i v e buoyant d e n s i t i e s i n cesium c h l o r i d e . The precedure i s d e t a i l e d I n " M a t e r i a l s and Methods" and the c a l c u l a t i o n s are set f o r t h i n Appendix I I . C e l l w a l l sugars were determined as o u t l i n e d i n " M a t e r i a l s and Methods" s e c t i o n . A l s o p l e a s e see F i g . 7 ( c e l l w a l l sugar chromatography) and accompanying discussion. Sugar + i n d i c a t e s l a r g e amounts of t h e p a r t i c u l a r sugar produced. P a t h o g e n i c i t y f o r mice was determined as d e s c r i b e d under V i r u l e n c e i n " M a t e r i a l s and Methods" s e c t i o n .  42  '  *0  , €' *  3jl ^  J *  fit  I-TVAS''  ^  »  /  * I  -  .  F i g . 6. S t r a i n s o f C. k u t s c h e r i which were used i n t h i s study. A, Wl; B, W2; both of which were i s o l a t e d i n from WSW mice. C, ATCC 1 5 6 7 7 ; D, presumed a v i r u l e n t v a r i a n t ATCC 15678. The l a t t e r two organisms were I s o l a t e d from the Lynch s t r a i n of Swiss white mice a t the R o c k e f e l l e r U n i versity. Gram s t a i n . (x 2 , 0 0 0 ) .  ,"\  A T C C 15677  ATCC  15678  P i g . 7. C e l l w a l l sugar chromatography. A c i d h y d r o l y s a t e s of C. k u t s c h e r l s t r a i n s ATCC I 5 6 7 7 and ATCC 1 5 6 7 8 were developed 24 h r s w i t h l s o p r o p y l : w a t e r : e t h y l acetate. The major sugars o f ATCC 1 5 6 7 7 a r e g a l a c t o s e and arabinose. The presumed v a r i a n t s t r a i n , ATCC 1 5 6 7 8 , has g a l a c t o s e and rhamnose, but no arabinose.  44  was measured as 42.0  ( c a l c u l a t e d i n Appendix  d i f f e r e n c e s had a c o n s i d e r a b l e  bearing  II).  These  on the d i r e c t i o n  taken by t h i s study, f o r i t was decided on the b a s i s o f the l a t t e r two experiments not to pursue a l i n e o f study which depended on the h y p o t h e s i s t h a t the v i r u l e n t ATCC  15678  the proposed a v i r u l e n t v a r i a n t ATCC to the o t h e r i n v i v o . are great two  15677  and  may transform one  That i s , the b i o c h e m i c a l  differences  enough t h a t even a c l o s e r e l a t i o n s h i p between the  s t r a i n s cannot be explained  i n terms o f g e n e r a l l y  accepted g e n e t i c p r i n c i p l e s . Pathogenicity  determination.  g e n i c i t y o f the v a r i o u s  The r e l a t i v e patho-  s t r a i n s of C. k u t s c h e r l used i n t h i s  study, as determined by intravenous i n j e c t i o n o f the organisms i n t o both l a t e n t l y i n f e c t e d and C. k u t s c h e r l f r e e mice, i s i l l u s t r a t e d by the r a t e o f s u r v i v a l o f i n f e c t e d mice as set f o r t h i n T a b l e s IV, V and VI. al  (34),  by Fauve et  the a v i r u l e n t v a r i a n t C. k u t s c h e r l ATCC  non pathogenic (Table ganisms.  As r e p o r t e d  IV) even i n doses of 10  15678 i s  viable or-  I t was a l s o non pathogenic f o r A / J and CF^(SPF)  mice i n j e c t e d i n the same way. a l l pathogenic.  The remaining s t r a i n s a r e  However, much l a r g e r numbers of the v i r u -  l e n t organism a r e r e q u i r e d  to i n f e c t the l a t e n t l y i n f e c t e d  mouse (WSW) as compared to the C. k u t s c h e r i f r e e mice, which are r e l a t i v e l y  susceptible.  the v i r u l e n c e of C. k u t s c h e r i  As shown i n T a b l e s V and VI, s t r a i n s ATCC  «  15677,  and s t r a i n s  45 TABLE IV S u r v i v a l of 8-12 wk o l d WSW Mice I n j e c t e d I n t r a v e n o u s l y w i t h Doses of V a r i o u s C. k u t s c h e r i s t r a i n s . Bacterial Strain  Dose  G.k. Wl  io3  C.k. W2  C.k. 1 5 6 7 7  •C.k. 1 5 6 7 8  No. Mice  Survivors  a t day  • •  2  4  7  14  6  6  6  6  6  105  12  12  12  12  11  10?  6  5  2  1  0  io3  6  6  6  6  6  105  12  12  12  11  10  107  6  4  1  0  0  103  6  6  6  6  6  12  12  12  12  12  10?  6  5  4  1  0  10?  12  12  12  12  12  A d u l t (8-12 wk o l d ) mice were used i n these s t u d i e s . The organisms were suspended i n 0.1 ml of Hanks' balanced s a l t s o l u t i o n which was i n j e c t e d i n t o a l a t e r a l t a i l v e i n of the r e c i p i e n t mouse. I n j e c t e d mice were housed 6 t o a cage and observed d a i l y f o r 14 days. The s u r v i v i n g mice were s a c r i f i c e d and a g r o s s i n s p e c t i o n performed a t the c o n c l u s i o n of the experiment. * C. k u t s c h e r l ATCC 15678 ( A v i r u l e n t v a r i a n t ) was l i k e w i s e i n j e c t e d i n t o 6 a d u l t CF-^SPF) and 6 a d u l t A / J mice i n doses-of 1 0 7 organisms and was a l s o found to be non-pathogenic f o r these s t r a i n s .  46  TABLE V S u r v i v a l of D i f f e r e n t Mouse S t r a i n s I n j e c t e d I n t r a v e n o u s l y w i t h V a r i o u s Doses o f C. k u t s c h e r i ATCC  15677  Mouse Strain  Dose  WSW  CF  X  A/J  No. Mice  2  4  7  14  io3  6  6  6  6  6  10*  12  12  12  12  12  10?  6  5  4  1  0  io3  6  6  6  5  3  12  11  7  4  0  10?  6  1  0  0  0  103  6  6  6  5  4  105  12  10  5  3  0  10?  6  2  1  1  0  P l e a s e see Table IV f o r o u t l i n e of experimental  procedure.  k?  TABLE VI S u r v i v a l o f D i f f e r e n t Mouse S t r a i n s I n j e c t e d Intravenously w i t h Various Doses: of C. k u t s c h e r i Wl  Mouse Strain  Dose  WSW  103  A/J  No. Mice  S u r v i v o r s at days 2  4  7  14  6  6  6  6  6  105  12  12  12  12  11  10?  6  5  2  1  0  103  6  6  4  4  3  105  12  10  5  3  0  10?  6  2  0  0  0  103  6  6  5  3  2  105  12  9  4  1  0  107  6  1  0  0  0  Please see Table IV f o r o u t l i n e of experimental  procedure.  48  F i g . 8. A gross c o r y n e b a c t e r i a l abscess l o c a t e d i n the mesenteric node o f a male WSW mouse s a c r i f i c e d 8 weeks a f t e r i n t r a p e r i t o n e a l i n j e c t i o n o f 10' C. k u t s c h e r i s t r a i n Wl. Symptoms Involved r u f f l e d f u r , a waddling g a i t and lethargy. The mouse was 8-12 weeks o l d when I n j e c t e d . The t o t a l weight on s a c r i f i c e was 26 g and the abscess weight was 9.8 g.  F i g . 9. A kidney which was removed from a male WSW mouse a f t e r death 14 days f o l l o w i n g Intravenous I n j e c t i o n of 10' C. k u t s c h e r l s t r a i n Wl. The mouse was 8-12- weeks o l d a t the time of i n j e c t i o n . The nodular abscesses i n the r e n a l c o r t e x c h a r a c t e r i s t i c a l l y develop when s u f f i c i e n t numbers o f organisms a r e i n j e c t e d i n t r a v e n o u s l y . I f smaller numbers a r e i n j e c t e d , the abscesses may appear and l a t e r regress.  F i g . 10. C. k u t s c h e r i i n a crushed nodule removed from the l i v e r of a male WSW mouse w i t h RLV induced coryneb a c t e r i a l pseudotuberculosis. The mouse was s a c r i f i c e d 28 days a f t e r I n j e c t i o n o f 50 I D 5 0 o f RLV as a weanling ( 4 - 5 weeks o l d ) . A c t i v a t i o n of l a t e n t c o r y n e b a c t e r i a l pseudot u b e r c u l o s i s i n WSW mice f r e q u e n t l y i n v o l v e d the l i v e r . Gram s t a i n . (x 2,000).  F i g . 11. C. k u t s c h e r l I n a n e c r o t i c area o f the r e n a l c o r t e x 14 days a f t e r intravenous i n j e c t i o n o f 10' C. k u t s c h e r i s t r a i n W2 i n t o an 8-12 week o l d male WSW mouse. Touch p r e p a r a t i o n , Giemsa s t a i n e d , (x 2,000).  5  0  F i g . 12. I n f i l t r a t i o n of small monocytic c e l l s i n t o n e c r o t i c l i v e r t i s s u e o f a mouse i n which c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s was induced by i n j e c t i o n o f 100 ID^Q o f RLV. The mouse was i n j e c t e d a t 8-12 weeks o f age and s a c r i f i c e d 28 days l a t e r . T h i s type of i n f i l t r a t e i s c h a r a c t e r i s t i c o f c h r o n i c i n f e c t i o n s such as t u b e r c u l o s i s . Note the d i s i n t e g r a t i n g p h a g o c y t i c c e l l a t bottom l e f t which i s shown r e l e a s i n g l a r g e numbers o f p r e v i o u s l y i n g e s t e d C. k u t s c h e r l . Gram s t a i n . (x 2,000).  51 Wl  and  W2  i s o l a t e d i n t h i s study, i s s i m i l a r .  venous i n j e c t i o n of 10$ or A/J mice was received  the  v i r u l e n t C. k u t s c h e r i  i n v a r i a b l y f a t a l , whereas WSW  same numbers of organisms u s u a l l y  T h i s confirms the o b s e r v a t i o n  into  induced are g e n e r a l l y  of l a r g e numbers of C. k u t s c h e r l .  intra-  CF (SPF) 1  mice which recovered.  by Pierce-Chase et a l  t h a t mice i n which the d i s e a s e c o r y n e b a c t e r i a l c u l o s i s can be  Thus,  (62)  pseudotuber-  r e s i s t a n t to i n j e c t i o n  I t was  considered probable  t h a t the answer to the q u e s t i o n of what makes c e r t a i n mouse s t r a i n s or c o l o n i e s , presumably l a t e n t l y i n f e c t e d ones, more r e s i s t a n t to exogenous i n f e c t i o n than o t h e r s would help e x p l a i n the  s t a t e of l a t e n t i n f e c t i o n and  why  c e r t a i n kinds  of p h y s i o l o g i c a l o r immunological s t r e s s induce a c t i v e disease.  I t seemed l o g i c a l t h a t the h a r b o r i n g of numbers of  C. k u t s c h e r i  i n o r on the l a t e n t l y i n f e c t e d mouse would  Induce s p e c i f i c antibody p r o d u c t i o n to a l e v e l h i g h enough to g i v e a l e v e l of p r o t e c t i o n a g a i n s t t h i s aspect i s d i s c u s s e d I t was instance  superinfection,  and  later.  a l s o noted i n t h i s experiment that i n no  d i d the d i s e a s e which r e s u l t e d from intravenous o r  intraperitoneal administration the n a t u r a l l y o c c u r r i n g , peritoneal  duplicate  the pathology of  o r induced d i s e a s e .  Thus i n t r a -  i n j e c t i o n r e s u l t e d i n the f o r m a t i o n of a l a r g e  abscess o r abscesses i n the mesenteric nodes ( F i g . 8) whereas intravenous I n j e c t i o n r e s u l t e d i n m u l t i p l e of the r e n a l c o r t e x  ( F i g . 9).  The  induced  nodular abscesses disease,  52 r e g a r d l e s s of whether RLV or c o r t i s o n e was  used as  i n d u c i n g agent, appeared as nodular o r c o n f l u e n t of the l i v e r , lungs,  spleen, o r b r a i n .  the  abscesses  These d i f f e r e n c e s  i n the development of the d i s e a s e must r e f l e c t the  dif-  f e r e n c e s i n the manner i n which the d i s e a s e i s i n i t i a t e d . For exogenous i n f e c t i o n of healthy mice to take p l a c e , between 10^ and IO'' organisms must be i n t r o d u c e d , abscess  and  f o r m a t i o n l a t e r takes p l a c e only i n areas where the  host defense mechanisms are unable to cope with the massive numbers trapped.  In a c t i v a t i o n of l a t e n c y , however, the  d i s e a s e e v i d e n t l y develops  i n areas i n which the organisms  appear to be r e a d i l y destroyed f o l l o w i n g i n j e c t i o n of l a r g e numbers i n t o healthy mice.  For example, e a r l y i n the i n -  duced d i s e a s e , numerous small f o c i of i n f e c t i o n may i n the l i v e r o r lung, and these nodular abscesses enlarge and l a t e r become c o n f l u e n t . from these  Two  appear  gradually  q u e s t i o n s emerge  observations:  1. What Is the immediate source of the organisms i n the l e s i o n s which develop f o l l o w i n g a c t i v a t i o n of l a t e n t i n f e c t i o n , harbored  i . e . , were they  i n small numbers i n the l i v e r or  lung, t h e r e to grow when a breakdown of host Immune system permitted?  the  The f a c t o r s  which m a i n t a i n o r Induce a c t i v a t i o n of l a t e n t i n f e c t i o n would be b e t t e r understood i t be known how  could  these organisms get i n t o  remain where they  are.  and  53 2. What a r e the changes which occur i n the l a t e n t l y i n f e c t e d host which suddenly  permit  C. k u t s c h e r i  to t h r i v e , and e s p e c i a l l y to t h r i v e i n areas where the organisms a r e a p p a r e n t l y w e l l cont r o l l e d i n healthy  animals?  O b s e r v a t i o n o f which f a c t o r s change d u r i n g the k i n d o f p h y s i o logical  s t r e s s capable of i n d u c i n g a c t i v a t i o n of l a t e n c y , and  which do not, could a t l e a s t e l i m i n a t e those f a c t o r s which are not r e s p o n s i b l e . of b a c t e r i a  For example, i f the r a t e o f c l e a r a n c e  from the c i r c u l a t i n g blood by the RES remains  as high d u r i n g the e a r l y e r y t h r o b l a s t i c stage o f the Rauscher d i s e a s e as i t i s i n normal mice, then r a t e of c l e a r a n c e i s e v i d e n t l y not a f a c t o r i n a c t i v a t i o n . I s o l a t i o n of G. k u t s c h e r l .  The r e s u l t s which a r e r e -  p o r t e d i n t h i s s e c t i o n d e a l o n l y with attempts to i s o l a t e C. k u t s c h e r i from a p p a r e n t l y h e a l t h y 8-12 week o l d WSW as they were r e c e i v e d from t h e departmental  stock.  mice  Thus,  the only s p e c i a l c o n d i t i o n which a p p l i e d to these mice i s t h a t they were presumed to be l a t e n t l y i n f e c t e d . C. k u t s c h e r l was i s o l a t e d from the t h r o a t i n o n l y 2 out of 100 attempts,  and not a t a l l from the s k i n and f u r  of the abdominal r e g i o n i n the same number of attempts. These f i n d i n g s are s i m i l a r t o those of o t h e r workers who have attempted t o I s o l a t e the organism from c u l t u r e s o f i n t e r n a l organs (7, 18, 3^» 80).  In view of the low  54 percentage of success,  and In c o n s i d e r a t i o n o f s i m i l a r  f i n d i n g s by o t h e r workers, i t could be argued t h a t ful  I s o l a t i o n s came only from those mice which were  disposed" and  success-,  i n some way.  subsequently  Another p o s s i b l e theory  discounted  "pre-  considered  by Fauve et a l (34) was t h a t  o n l y a small percentage of mice a r e l a t e n t l y i n f e c t e d , and the d i s e a s e i s spread are under s t r e s s .  from mouse to mouse i n groups which  T h i s aspect  i s considered b r i e f l y under  C o r t i s o n e s t u d i e s i n t h i s s e c t i o n and more f u l l y  i n the  general d i s c u s s i o n . Latency  t r a n s f e r attempts.  I n these  attempts were made t o t r a n s f e r l a t e n c y from  experiments, apparently  h e a l t h y 8 - 1 2 week o l d WSW mice ( l a t e n t l y i n f e c t e d ) from the departmental stock, t o groups of imported A / J and CF^(SPF) mice t h a t were C. k u t s c h e r l f r e e .  The means of t r a n s f e r  attempted was I n j e c t i o n of organ homogenates from WSW (as p r e v i o u s l y d e s c r i b e d ) . was contingent  mice  The success of the experiment  on a l l o r a heavy m a j o r i t y of WSW mice being  i n f e c t e d , on a s u f f i c i e n t q u a n t i t y o f a p p r o p r i a t e t i s s u e being t r a n s f e r r e d , on a means o f a c t i v a t i n g the newly a c q u i r e d l a t e n t i n f e c t i o n and on a s u f f i c i e n t number o f mice being used. Fauve et a l (34) have concluded tend to be l a t e n t l y I n f e c t e d .  that e n t i r e colonies  I n t h i s experiment a t o t a l  of 24 randomly s e l e c t e d donor mice were used so t h a t even if  the e n t i r e colony was not l a t e n t l y i n f e c t e d , chances o f  55 o b t a i n i n g many C. k u t s c h e r l f r e e mice were remote.  The  t i s s u e homogenate s u p p l i e d approximately 10 mg of s p l e e n and 50 mg of l i v e r mouse.  from the donor mouse to each r e c i p i e n t  (Where i n f e c t i o n i s induced i n WSW  mice i t most  f r e q u e n t l y appears i n the l i v e r of the a f f e c t e d a n i m a l ) . Finally,  c o r t i s o n e a c t i v a t i o n has been found t o be  s u c c e s s f u l i n WSW  mice.  Of 72 r e c i p i e n t mice,  20%  assuming  s u c c e s s f u l t r a n s f e r , 14 animals might be expected to respond to c o r t i s o n e treatment. G. k u t s c h e r i could not be i s o l a t e d from any of the r e c i p i e n t mice 21 days a f t e r c o r t i s o n e treatment.  At the  c o n c l u s i o n of the experiment, a mucoid a s c i t e s f l u i d ( F i g . 14) was mice. fluid cells,  found i n the peritoneum of many o f the r e c i p i e n t Examination o f Giemsa and Gram s t a i n e d smears of t h i s  showed t i s s u e d e b r i s and l a r g e numbers of monocytic but no b a c t e r i a .  The organs of these mice presented  no s i g n of c o r y n e b a c t e r i a l i n f e c t i o n , nor could C. k u t s c h e r l be i s o l a t e d from any of them. I t c o u l d be contended that the c o n d i t i o n s o f  the  experiment do not exclude the p o s s i b i l i t y of s u c c e s s f u l l y demonstrated t r a n s f e r o f l a t e n c y if,  i f more mice were used o r  f o r example, c o r t i s o n e treatment had been extended f o r  another t h r e e o r s i x weeks.  Such an argument makes the  apparent tendency of whole c o l o n i e s to become l a t e n t l y i n f e c t e d by n a t u r a l mouse to mouse contact d i f f i c u l t stand.  T h i s aspect o f the p r e s e n t study was  to under-  t h e r e f o r e not  56 extended, and  i t was  concluded  t h a t l f C. k u t s c h e r l i s  present i n the l i v e r or s p l e e n of l a t e n t l y i n f e c t e d it  i s present i n extremely  low numbers.  Cortisone administration. sone a c e t a t e was latent  mice,  I n j e c t i o n of h y d r o c o r t i -  used i n t h i s study as a means of  verifying  corynebacterial pseudotuberculosis i n representative  groups of WSW  mice and  c o n v e r s e l y , as a means of demon-  s t r a t i n g t h a t the Imported s t r a i n s of mice (CF-^SPF) and A/J) were, as r e p o r t e d , C. k u t s c h e r l f r e e . garding s t r a i n , age,  The d e t a i l s r e -  sex, numbers of mice, a d m i n i s t r a t i o n of  F i g . 13. N e c r o t i c l i v e r of a male WSW mouse which developed c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s f o l l o w i n g i n j e c t i o n of 12.5 mg of h y d r o c o r t i s o n e a c e t a t e a t 8-12 weeks of age. The mouse had r u f f l e d f u r and a waddling g a i t , and was s a c r i f i c e d a t day 2 1 . The pseudotuberculos i s induced by RLV resembles the c o r t i s o n e Induced d i s ease, but i n j e c t i n g v i r u l e n t C. k u t s c h e r l i n t o healthy mice does not d u p l i c a t e the induced i n f e c t i o n .  57  F i g . 14. C e l l s i n mucoid a s c i t e s o f A / J mouse which was i n j e c t e d i n t r a p e r i t o n e a l l y w i t h l i v e r and s p l e e n homogenates from WSW mice (see Latency t r a n s f e r attempts). C o r t i s o n e was a d m i n i s t e r e d a t time of i n j e c t i o n , f l u i d was taken a t 21 days. No C. k u t s c h e r i were observed. A, c e l l s , predominantly small lymphocytes, x 200; B, same p r e p a r a t i o n w i t h d i f f e r e n t types of c e l l s p r e s e n t . P a l e c e l l s a r e e r y t h r o c y t e s , very dark c e l l s a r e small lymphocytes.  Giemsa s t a i n .  (x 2,000).  58  TABLE V I I The E f f e c t o f C o r t i s o n e A d m i n i s t r a t i o n on the Incidence of C o r y n e b a c t e r i a l P s e u d o t u b e r c u l o s i s o r t h e i s o l a t i o n of C. k u t s c h e r i from the Organs of V a r i o u s Mouse S t r a i n s  Mouse Strain  WSW  No. of Mice 48  S i n g l e Dose (12.5 mg) C.k. Deaths isolated  5  9  24  CP (SPF) 1  12  1  8 8  0  7  5  1  0  0  0  0  12  A/J  D i v i d e d Dose (5 x 2 . 5 mg) C.k. Deaths i s o l a t e d  0  Mice used were a d u l t males ( 8 - 1 2 weeks) weighing a p p r o x i mately 25 g. Deaths were recorded up to t h e 2 1 s t day a f t e r the f i r s t h y d r o c o r t i s o n e a c e t a t e i n j e c t i o n . In the f i r s t group, 12 mice were housed s e p a r a t e l y , and o f t h i s number C. k u t s c h e r i were i s o l a t e d from 3 . A H o t h e r mice were housed not more than 6 to a cage, w i t h CF.(SPF) and A / J mice maintained i n i s o c a g e s . Gross examinations were made o f dead animals where p o s s i b l e . I s o l a t i o n s of C. k u t s c h e r i were made a s d e s c r i b e d e a r l i e r .  59 the drug and animal housing appear as p a r t of Table V I I , where the e f f e c t s on the t h r e e mouse s t r a i n s used and the e f f e c t of s i n g l e v e r s u s m u l t i p l e dosage o f c o r t i s o n e are compared. G. k u t s c h e r l could be i s o l a t e d from the c o r t i s o n e t r e a t e d WSW  mice w i t h a frequency of about 20%, but the  organisms were never i s o l a t e d from A/J o r C F ^ S P F ) s t r a i n s . Further,  i n c i d e n c e of c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s i n  mice housed i n d i v i d u a l l y matched approximately the i n animals housed 6 to a cage.  incidence  The l a t t e r o b s e r v a t i o n i s  c o n s i s t e n t w i t h the r e p o r t o f Fauve et a l (3*0  t h a t the  d i s e a s e i s not spread s i g n i f i c a n t l y from mouse to mouse d u r i n g the a c t i v a t i o n procedure.  I t was  b a s i s of these experiments that WSW  concluded on the  mice are l a t e n t l y i n -  f e c t e d and A/J and CF-^SPF) mice are not. When 1 2 . 5  mg of c o r t i s o n e was  dose, r e s i d u a l c o r t i s o n e was  i n j e c t e d as a s i n g l e  sometimes found i n the p e r i -  t o n e a l c a v i t y 3 weeks l a t e r , an i n d i c a t i o n t h a t the e f f e c t of the massive dose p e r s i s t e d d u r i n g the e n t i r e course o f the experiment.  There appeared to be no advantage,  there-  f o r e , to a d m i n i s t r a t i o n of the d i v i d e d dosage, which a l s o i n v o l v e d a h i g h e r r i s k of i n t r o d u c i n g microorganisms a l o n g the t r a c k of the needle.  (G. k u t s c h e r i i n a c t i v a t i o n o f  l a t e n c y was always found i n i n t e r n a l organs and never observed- a t the s i t e of i n j e c t i o n , a l t h o u g h o t h e r organisms, e.g., s t a p h y l o c o c c i , were sometimes found i n abscesses which  6o formed a t the s i t e where the s k i n had been broken by the needle). Where a c t i v e c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s developed as a r e s u l t o f c o r t i s o n e a d m i n i s t r a t i o n , abscesses appeared  as m u l t i p l e nodules,., becoming c o n f l u e n t i n l a t e r  stages, i n the l i v e r , l u n g s and spleen. the c o r t i s o n e induced d i s e a s e resembles  I n t h i s regard, the RLV induced  c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s i n t h a t the l e s i o n s do not have the gross appearance o r l o c a t i o n o f the gross abscesses which develop a f t e r i n j e c t i o n o f v i r u l e n t G. kuts c h e r l organisms.  T y p i c a l l i v e r l e s i o n s i n the induced  disease are i l l u s t r a t e d i n F i g . 13.  The r e s u l t s  observed  are s i m i l a r to those of o t h e r workers ( 3 , 3^« 49). Macrophage s t u d i e s .  As shown i n Table V I I I , the  p h a g o c y t i c index f o r i n g e s t i o n of heat k i l l e d  C. k u t s c h e r l  s t r a i n Wl b a c t e r i a was s i g n i f i c a n t l y i n c r e a s e d (from 1 . 0 0 to 1.89) when pooled s e r a from WSW mice was used CF^(SPF) s e r a from mice of approximately ever, when the WSW  i n place of  the same age.  How-  s e r a was harvested from mice i n f e c t e d  w i t h RLV 14 days p r e v i o u s l y , the improvement i n the r a t e o f p h a g o c y t o s i s was l o s t apparent  (from 1.89 down to 1 . 0 5 ) .  It i s  t h a t f a c t o r s a r e present i n the s e r a of the l a t e n t l y  i n f e c t e d WSW mouse t h a t a r e absent  i n the C. k u t s c h e r l f r e e  C F ^ S P F ) mouse. Since a c t i v a t i o n o f l a t e n t c o r y n e b a c t e r i a l pseudot u b e r c u l o s i s e v i d e n t l y i s I n i t i a t e d i n the e a r l y days o r  61 weeks o f the Rauscher d i s e a s e , the drop  i n the p h a g o c y t i c  index when c e l l s a r e supplemented with the sera harvested from RLV i n f e c t e d mice i s of s p e c i a l i n t e r e s t . tempting  It i s  to conclude t h a t the WSW mouse has developed  k u t s c h e r i s p e c i f i c antibody a t some time d u r i n g the course o f the l a t e n t  C.  (or c o n t i n u o u s l y )  I n f e c t i o n , and t h a t t h i s  antibody l e v e l drops as the Rauscher d i s e a s e p r o g r e s s e s . There i s s t r o n g evidence, however, t h a t e x i s t i n g l e v e l s of antibody do not drop d u r i n g the course of the. Rauscher d i s ease ( 3 5 . 7 4 ) .  Furthermore,  p a s s i v e hemagglutination,  which  was shown i n t h i s study to d e t e c t C. k u t s c h e r i s p e c i f i c antibody i n immune s e r a a t a d i l u t i o n o f l : 5 i l 2 0 ,  d i d not  demonstrate any c i r c u l a t i n g antibody i n normal WSW  sera.  One e x p l a n a t i o n c o u l d be t h a t the t i t e r o f c i r c u l a t i n g body i s too low i n normal WSW hemagglutination, cytosis.  sera to be d e t e c t e d by p a s s i v e  and y e t h i g h enough to s t i m u l a t e phago-  A t such a low t i t e r , a very small decrease d u r i n g  the course of RLV i n f e c t i o n " (due to the constant of  anti-  C. k u t s c h e r l organisms) would exhaust  the supply of a n t i -  body and the r a t e of p h a g o c y t o s i s would drop. possibility  presence  A remote  i s t h a t one o f the e f f e c t s of the Rauscher d i s -  ease i s the p r o d u c t i o n o f f a c t o r s i n h i b i t o r y t o phagocytosis, which accumulate i n the s e r a d u r i n g the d i s e a s e . p o s s i b i l i t y i s t h a t the f a c t o r s i n WSW  Another  serum r e s p o n s i b l e f o r  i n c r e a s e d p h a g o c y t o s i s are not immunospecific.  Some o f the  components o f complement a r e q u i t e u n s t a b l e and would soon  TABLE V I I I In v i t r o p h a g o c y t o s i s of C. k u t s c h e r i by WSW p e r i t o n e a l macrophage i n the presence of pooled mouse s e r a from d i f f e r e n t sources. Source of Pooled Sera  C o n d i t i o n of Mouse  Phagocytic Index  CF (SPP)  Normal (C. k u t s c h e r i f r e e )  1.00  WSW  Normal (Latent p s e u d o t u b e r c u l o s i s )  1,89  WSW  RLV i n f e c t e d (Latent p s e u d o t u b e r c u l o s i s )  1<05  1  The organism used i n t h i s experiment was heat k i l l e d C . k u t s c h e r l s t r a i n Wl. The experimental procedure used i n h a n d l i n g the c u l t u r e i s o u t l i n e d ' i n the c a p t i o n accompanying F i g . 1 5 . Sera was c o l l e c t e d from the pooled blood of 10 mice f o r each p o o l , and was heated to 5 ^ C f o r 10 min b e f o r e use. The c a l c u l a t i o n s of the p h a g o c y t i c index d e t e r m i n a t i o n are d e t a i l e d i n Appendix I I I . B r i e f l y , the t o t a l b a c t e r i a i n g e s t e d by the f i r s t 50 phagocytosing c e l l s were counted f o r each p r e p a r a t i o n observed. The v a l u e s f o r a l l p r e p a r a t i o n s were a d j u s t e d so t h a t the index expressed f o r p h a g o c y t o s i s w i t h normal serum supplement, C F n ( S P F ) , was equal to 1.00. At l e a s t 9 p r e p a r a t i o n s were observed and were averaged f o r each of the 3 s e r a .  63  F i g . 15. Mouse p e r i t o n e a l macrophages, m a i n t a i n e d under t i s s u e c u l t u r e c o n d i t i o n s , which have "been f i x e d and s t a i n e d a f t e r i n g e s t i o n o f heat k i l l e d C. k u t s c h e r l s t r a i n Wl organisms i n v i t r o . 1 ml of E a g l e ' s minimal medium , supplemented w i t h 10$ f e t a l c a l f serum and c o n t a i n i n g 10 cashed CF]_(SPF) mouse p e r i t o n e a l macrophage (and o t h e r c e l l s ) was p i p e t t e d i n t o L e i g h t o n tubes c o n t a i n i n g cover s l i p s , t i g h t l y stoppered, and i n c u b a t e d a t 37C. During two subsequent medium changes, c e l l s o t h e r than macrophages were washed away; w h i l e macrophage spread out and adhered t o t h e g l a s s . F i n a l l y , t h e medium c o n t a i n i n g f e t a l c a l f serum supplement was r e p l a c e d by the same medium supplemented w i t h 10$ o f the p o o l e d mouse s e r a under t e s t , which a l s o c o n t a i n e d heat k i l l e d C. k u t s c h e r l a t a c o n c e n t r a t i o n o f 2 x 10? c e l l s ml. I n c u b a t i o n was c o n t i n u e d f o r 30 min w i t h g e n t l e a g i t a t i o n every 5 min. The c o v e r s l i p s t h e n were removed, r i n s e d i n Hanks' s o l u t i o n , f i x e d i n ether:95$ e t h a n o l 1:1 and Glemsa s t a i n e d ,  (x 2,000).  64 be  exhausted u n l e s s  one  continuously  produced (44).  Perhaps  o r more of these f a c t o r s becomes exhausted d u r i n g  the  e r y t h r o b l a s t i c stage of the Rauscher d i s e a s e . Immunization and  serology.  A preliminary  experiment  which was  designed to determine whether C. k u t s c h e r i  vaccine  conferred  s i g n i f i c a n t immunity to t h i s organism, was  carried  out  e a r l y i n t h i s study on 12 a d u l t male WSW  mice.  These  mice were I n j e c t e d i n t r a p e r i t o n e a l l y w i t h h e a t - k i l l e d C. k u t s c h e r l s t r a i n Wl v a c c i n e prepared and described  i n the M a t e r i a l s and  Methods s e c t i o n .  no evidence that t h i s treatment c o n f e r r e d t e c t i o n on challenge  administered There  The  of v i r u l e n t C. k u t s c h e r l s t r a i n  intravenously.  challenge  dose was  10?  These two  organisms i n j e c t e d  were s a c r i f i c e d and  two  gross i n -  s p e c t i o n r e v e a l e d m u l t i p l e abscesses i n the r e n a l I t was  cortex.  concluded t h a t the organisms were not h i g h l y  genic and  that i n j e c t i o n of v a c c i n e  Wl was  S i x mice d i e d w i t h i n three days and  s u r v i v e d 14 days.  was  s i g n i f i c a n t pro-  organisms, 10 days a f t e r the immunization programme completed.  as  d i d not  confer  antiprotec-  t i o n of a s i g n i f i c a n t l y higher l e v e l than the n a t u r a l l e v e l of r e s i s t a n c e that WSW  mice have to these organisms.  t h e r i n v e s t i g a t i o n of t h i s aspect was  Fur-  t h e r e f o r e not c a r r i e d  out. Passive  h e m a g g l u t i n a t i o n was  subsequently e s t a b l i s h e d  as being a s e n s i t i v e method f o r d e t e c t i n g  circulating  65 C. k u t s c h e r i antibody i n mice.  The pooled immune s e r a of  12  a d u l t CF]_(SPF) mice showed hemagglutination a t a d i l u t i o n  of  1:5,120.  Pooled s e r a of twelve a d u l t WSW  mice on the  same immunization programme had the same t i t e r .  Thus, both  C. k u t s c h e r i f r e e and l a t e n t l y i n f e c t e d mice can produce  a  h i g h l e v e l of c i r c u l a t i n g antibody i n response to i n j e c t i o n of  heat k i l l e d  C. k u t s c h e r i  organisms.  In a separate experiment, weanling WSW  pooled s e r a of immunized  mice which r e c e i v e d 50  ID50  °f  seven days  p r e v i o u s to the f i r s t  C. k u t s c h e r l v a c c i n e i n j e c t i o n showed  a t i t e r of o n l y l 6 0 .  Because s e r a were not added to t h i s  p o o l u n l e s s the donor mouse had a r e t i c u l o c y t e count i n excess of 3.4 a t day 14,  i t was  assumed t h a t a l l s e r a came from  mice i n the e a r l y o r e r y t h r o b l a s t i c stage of the Rauscher disease.  I n d i v i d u a l t i t r a t i o n s of 6 weanling mice immunized  i n an i d e n t i c a l manner showed that 3 d i d not produce a b l e c i r c u l a t i n g antibody.  Splenomegaly and  titrat-  retlculocytosis  were p r e s e n t i n a l l mice i n which antibody s y n t h e s i s was pressed.  One  de-  mouse w i t h a t i t e r of 2 , 5 6 0 d i d not have an  enlarged s p l e e n ( l e s s than JQO mg weight) and a second, w i t h , a t i t e r of 3 2 0 ,  had developed p s e u d o t u b e r c u l o s i s d u r i n g the  course of the experiment  (the C. k u t s c h e r l organisms  isolated  from a l e s i o n were a d i f f e r e n t s t r a i n (Wl) from that used i n the v a c c i n e (ATCC 1 5 6 7 7 ) .  Pooled s e r a of 6 c o n t r o l s which  d i f f e r e d o n l y i n t h a t they r e c e i v e d no RLV t i t e r of 2 , 5 6 0 .  i n j e c t i o n , had a  The d e t a i l s of t h i s experiment  a r e set f o r t h  6  6  i n Table IX. A second method by which c i r c u l a t i n g antibody was demonstrated i n the sera o f immunized mice was i n d i r e c t immunofluorescent s t a i n i n g ( F i g . 1 6 ) .  A l l stains of v i r u -  l e n t C. k u t s c h e r l demonstrated f l u o r e s c e n c e G. kutscherl s t r a i n ATCC  1 5 6 7 7  antiserum o r s t r a i n Wl a n t i -  serum was used i n the procedure.  C o n t r o l s t r e a t e d w i t h the  serum o f normal mice d i d not f l u o r e s c e Absorption  when e i t h e r  significantly.  of immune serum w i t h C. k u t s c h e r l c e l l s e f f e c -  t i v e l y quenched the f l u o r e s c e n c e .  F i g . 1 6 . I n d i r e c t method o f immunofluorescent s t a i n i n g of C. k u t s c h e r l s t r a i n Wl c e l l s . The Immune mouse serum used was produced i n response to C. k u t s c h e r l ATCC 1 5 6 7 7 heat k i l l e d v a c c i n e and had a p a s s i v e hemagglutination t i t e r of 5 t l 2 0 . A f t e r treatment o f the smear of washed b a c t e r i a l c e l l s with the immune mouse serum, f l u o r e s c e i n conjugated goat anti-mouse Yglobulin was a p p l i e d . Cont r o l s t r e a t e d w i t h normal WSW mouse serum d i d not f l u o r e s c e significantly. (x 7 5 0 ) .  67  TABLE IX P a s s i v e Hemagglutination  Strain of Mouse  No. of mice i n serum pool  CF (SPF)  6  CF^SPF)  12  1  WSW  6  WSW  12 #1 #2 #3 #4 * #5 #6  WSW  1 1 1 1 1 1  WSW  17  WSW  6  Condition of Mouse  Spleen weight (day 21)  Titers Reticulocyte count (day 14)  Antibody titer (day 21)  -  normal  -  -  none  immunized  -  -  5.120  normal immunized  594 585 50 ID50 of RLV 524 a t day 0 , 175 C. k u t s c h e r l 500 2,606 vaccine at days 7 and 10  -  No RLV, but v a c c i n e at days 7 & 10  -  3.1 3.9 12.0 2.8 5.1 4.8 VIA.  -  none 5.120  none 80 none 2,560 320 none 160 2,560  * C o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s developed. P a s s i v e h e m a g g l u t i n a t i o n was performed w i t h tanned sheep e r y t h r o c y t e s which were coated w i t h C. k u t s c h e r l a n t i g e n as d e s c r i b e d under Serology i n " M a t e r i a l s and Methods".  68 Clearance s t u d i e s .  P r e v i o u s immunization  greatly  improves the c l e a r a n c e of the s p e c i f i c a n t i g e n from the c i r c u l a t i n g blood by c e l l s o f the r e t i c u l o e n d o t h e l i a l However, the maximum K f o r mice .(K = l o g C^ - l o g i s approximately  system.  02/^2-^1)»  independent  of antibody t i t e r ,  The average  K of 0.40 c a l c u l a t e d f o r C. k u t s c h e r l i n non-  immune a d u l t WSW mice i s t h e r e f o r e remarkably  0.40 ( 4 ) .  high.  It  c o u l d i n d i c a t e e i t h e r p r e v i o u s exposure o f the animal to C. k u t s c h e r l o r some p h y s i c a l o r chemical c h a r a c t e r i s t i c o f the organism  which a i d e d p h a g o c y t o s i s i n a  non-immunospecific  manner. Although  i t has been noted t h a t i t was not p o s s i b l e  to  d e t e c t C. k u t s c h e r i s p e c i f i c antibody i n the pooled s e r a ,  of  non immunized WSW mice by u s i n g the technique of p a s s i v e  hemagglutination,  t h i s same sera ( i n a 1:10 d i l u t i o n )  n e v e r t h e l e s s improved p h a g o c y t o s i s o f C. k u t s c h e r l by CF-j_(SPF) mouse p e r i t o n e a l macrophage i n v i t r o by almost 100$.  I t i s p o s s i b l e t h a t e i t h e r n o n - s p e c i f i c serum f a c t o r s  are i n v o l v e d , o r an antibody t i t e r too low t o be d e t e c t e d by p a s s i v e hemagglutination i s n e v e r t h e l e s s high enough to s i g nificantly The  improve p h a g o c y t o s i s , both i n v i t r o and i n v i v o .  c l e a r a n c e constant K of 0.28 observed  i n RLV i n f e c t e d  WSW mice would then be c o n s i s t e n t w i t h the l a c k of improvement i n p h a g o c y t i c index when s e r a from RLV i n f e c t e d mice was s u b s t i t u t e d f o r normal WSW p h a g o c y t o s i s experiments.  WSW  s e r a f o r the I n v i t r o  The K of 0.35 i n A / J mice, which  69  F i g . 17. Clearance constants, K, i n d i c a t i n g phagoc y t i c index of the r e t i c u l o e n d o t h e l i a l system f o r k i l l e d C. k u t s c h e r l organisms, f o r RLV i n f e c t e d WSW mice, f o r normal A/J mice and f o r normal WSW mice. The "bacteria were l a b e l e d w i t h P32 and I n j e c t e d i n t o a l a t e r a l t a i l v e i n and the r a d i o a c t i v i t y i n the c i r c u l a t i n g blood counted at intervals. Supporting d a t a and c a l c u l a t i o n s a r e i n c l u d e d i n Appendix IV;,  70 which i s r e a l l y not observed i n WSW  s i g n i f i c a n t l y d i f f e r e n t from the  mice, i s d i f f i c u l t  immunological standpoint,  unless  to i n t e r p r e t from  0.41  to 0.28  an  i t be allowed that  serum f a c t o r s i n v o l v e d i n c l e a r a n c e immunospecific.  0.40  by the RES  are  the not  I t i s concluded that the drop i n K from  f o r clearance  o f C. k u t s c h e r l by the RES  under  the c o n d i t i o n s where c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s activated i s significant,  even though the K of 0.28  is  is still  high. E l e c t r o n microscopy. terial  E l e c t r o n micrographs of bac-  c o l o n i e s demonstrated m u l t i n u c l e a t e d  phism and  heavy c e l l w a l l s  cells,  pleomor-  ( F i g . 18A.). I t might appear t h a t  the d e n s i t y of the c e l l w a l l could a f f o r d the microorganism some p r o t e c t i o n a g a i n s t  the c y t o l y t i c enzymes of the phago-  c y t o s i n g c e l l s of the host. could be advanced as one the organism i n host contributed latency. cortex,  Thus, b a c t e r i a l r e s i s t a n c e ,  explanation  t i s s u e , and  to the maintenance of  d e b r i s i n a l e s i o n of the r e n a l  14 days f o l l o w i n g intravenous  C. k u t s c h e r i s t r a i n Wl  a d m i n i s t r a t i o n of  i n t o an a d u l t male WSW  l a r g e numbers of C. k u t s c h e r l e x t r a c e l l u l a r l y . C. k u t s c h e r i i n the same p r e p a r a t i o n  In F i g .  scarce  18C,  i s p l a i n l y undergoing  Although these c e l l s are t h e r e f o r e able to  C. k u t s c h e r l , they are  10?  mouse, shows  d i s i n t e g r a t i o n w i t h i n v a c u o l e s of polymorphonuclear cytes.  of  would t h e r e f o r e be a f a c t o r  by the p a r a s i t e i t s e l f  In F i g . 18B,  f o r the p e r s i s t e n c e  leukodestroy  i n e s t a b l i s h e d l e s i o n s , where  71  F i g . 18. E l e c t r o n micrographs of C. k u t s c h e r i . A, from colony on blood agar, showing pleomorphism and heavy c e l l w a l l s t r u c t u r e (x 12,000); B, i n d e b r i s o f i n f e c t e d mouse kidney (x 5»000); C, a f t e r i n g e s t i o n by polymorphonuclear l e u k o c y t e s i n kidney, showing c y t o l y s i s (x 8,000). Phosphate b u f f e r e d g l u t a r a l d e h y d e and osmium f i x a t i o n , u r a n y l a c e t a t e and l e a d c i t r a t e s t a i n i n g .  72 small monocytic c e l l s and o c c a s i o n a l macrophages are found (Fig.  12).  I t has been shown by Fauve e t a l ( 3 2 ) that the  organism i s able to s u r v i v e and to m u l t i p l y i n s i d e macrophages.  P h a g o c y t o s i s by these c e l l s c o u l d t h e r e f o r e serve  to d i s s e m i n a t e the i n f e c t i o n .  The low t o x i c i t y  o f the  organism would permit l e n g t h y s u r v i v a l of the phagocytoslng c e l l and thus a i d f u r t h e r i n d i s s e m i n a t i o n and the maintenance o f l a t e n t I n f e c t i o n i f and when acute l e s i o n s regress.  73 Discussion Rauscher leukemia v i r u s (RLV). was  initially  The Rauscher v i r u s  i s o l a t e d from BALB/c mice i n which c e l l s (651.  o r i g i n a t i n g from a lymphoblastoma were being passaged The v i r u s i n f e c t s and of mice.  i s r e a d i l y passaged i n most s t r a i n s  As o r i g i n a l l y r e p o r t e d by Rauscher and  l a t e d upon by Dunn and Green ( 2 7 ) , disease:  extrapo-  RLV produces a dual-type  an e a r l y e r y t h r o b l a s t i c r e a c t i o n which i s f o l l o w e d  i n s u r v i v i n g mice by a t r a n s p l a t a b l e lymphoid  leukemia.  Because the presumed I n d u c t i o n of l a t e n t c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s must occur v e r y e a r l y i n the e r y t h r o b l a s t i c stage of the Rauscher d i s e a s e , t h i s stage r e c e i v e d the p r i n c i p a l c o n s i d e r a t i o n i n t h i s In BALB/c mice (6-5)  has  study.  the s p l e e n begins to enlarge  between 7 and 14 days a f t e r i n f e c t i o n , due  to the r a p i d  p r o l i f e r a t i o n o f e r y t h r o b l a s t i c c e l l s i n the red pulp, which soon r e p l a c e the normal r e d pulp. enormous ( F i g . 2 and and hemorrhage.  The  s p l e e n becomes  3) and death o f t e n o c c u r s from r u p t u r e  F o c i of e r y t h r o p o i e s i s appear i n l i v e r  somewhat l a t e r t h a n t h e i r appearance i n the spleen.  In- -  creased numbers of r e t i c u l o c y t e s and t e r m i n a l l y n u c l e a t e d r e d c e l l s are the f i r s t abnormality blood  ( F i g . 4).  a t a normal r a t e .  noted  i n the p e r i p h e r a l  These c e l l s accumulate and f a i l S i e g e l et a l ( 7 6 ) ,  BALB/c mice and i n j e c t i n g RLV  to mature  working w i t h  of h i g h t i t e r ,  weanling  detected^an  74 increase days.  i n c i r c u l a t i n g r e t i c u l o c y t e s as e a r l y as  They r e p o r t e d  i n nucleated  seven  r e t l c u l o c y t o s i s , anaemia and  increase  c e l l count reached a maximum i n s i x weeks.  lymphocytic leukemia was  reported,  presumably because  No  the  s e v e r i t y of the e r y t h r o b l a s t i c r e a c t i o n r e s u l t e d i n e a r l y death. I f the  spleen  i s removed, the e r y t h r o b l a s t i c f o c i  appear i n the l i v e r and  lymph nodes, but the  e r y t h r o b l a s t i c stage i s diminished  s e v e r i t y of  and most mice s u r v i v e ,  succumb to the lymphocytic leukemia which f o l l o w s . but  lymphocytosis  and  6-8  weeks a f t e r  monocytosis are not prominent u n t i l Dunn and  Green (27)  do not b e l i e v e the  f e r a t i n g c e l l i s a neoplastic reticulum  c e l l , but  to  Some  s t i m u l a t i o n of o t h e r c e l l s a l s o occurs,  infection.  the  prolian  e r y t h r o p o i e t i c c e l l i n d i s t i n g u i s h a b l e from c e l l s found i n the  spleens of normal mice s t i m u l a t e d  cytes. vival  to produce  Brodsky et a l (14) r e p o r t a 50$ r e d u c t i o n of 14 day  Rauscher d i s e a s e  red blood c e l l s .  claim that e r y t h r o p o i e s i s recovers Green ( 2 7 )  Dunn and  i n about 21  warned that RLV  a uniform e f f e c t even when g i v e n to the  on the  They a l s o  does not produce  same i n b r e d mice. and  the  i n f e c t e d animals which e v i d e n t l y depended  source of the v i r u s , the amount given,  of the mice r e c e i v i n g i t . be used i n RLV  i n sur-  days.  They found s i g n i f i c a n t d i f f e r e n c e s i n i n f e c t i v i t y pathology of the  erythro-  studies.  They urge that only  and  the  age  Inbred mice  75 Although t h i s p o i n t i s w e l l taken, i t i s not Rauscher d i s e a s e per periments, but and  se that i s being  a s t r a i n of mice known to be infection.  s t u d i e d i n these  i t s a c t i v i t y as a general  the r e s u l t s of such suppression  the ex-  immunosuppressant  of the immune system of  c a r r i e r s of a l a t e n t b a c t e r i a l  I t i s a c t u a l l y probable that an i d e n t i c a l  con-  d i t i o n of l a t e n c y would not be d u p l i c a t e d by i n j e c t i n g organisms i n t o an inbred s t r a i n of s p e c i f i c pathogen f r e e mice.  One  would r i s k p r o g r e s s i v e  disease,  or immune r e -  sponses that d i f f e r e d from the n a t u r a l l y a c q u i r e d One  of the d i f f i c u l t i e s i n working w i t h the WSW  the wide v a r i a t i o n i n the mice.  spleen  s i z e i n normal  latency.  mouse  was  (control)  Thus, some "normal" spleens were e x c i s e d which weighed  w e l l over 300  mg.  The  immunosuppressive e f f e c t of RLV  be considered  i n a l a t e r s e c t i o n , and  to the e f f e c t s of other  compared and  will  contrasted  chemical and p h y s i c a l phenomena which  have a l s o been observed to induce c o r y n e b a c t e r i a l pseudotuberculosis. Corynebacterial  pseudotuberculosis.  of c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s Kutscher i n 1894,  who  was  The  first  made by F r i e d r l c h  i s o l a t e d and d e s c r i b e d  a diphtheroid  as the c a u s a t i v e agent of a t u b e r c u l o s i s - l i k e d i s e a s e mice. murium.  He  named the organism B a c i l l u s I t was  bacterium.  report  in  pseudotuberculosis  a l s o once c l a s s i f i e d i n the genus Myco-  Bergey's Manual of Determinative  Bacteriology  76 (6)  c a l l s the e t i o l o g i c agent  Corynebacterium  although the names Corynebacterlum murium ( 3 ,  11,  63,  72)  and  are i n r e c e n t l i t e r a t u r e .  kutscherl,  pseudotuberculosis murium ( 7 .  Corynebacterlum The organism  i s an  irregularly  s t a i n i n g Gram p o s i t i v e rod, f r e q u e n t l y curved and w i t h s w o l l e n ends, with a s t r o n g tendency  48)  sometimes  to grow i n  c l u s t e r s ( F i g . 6.). When the d i s e a s e occurs n a t u r a l l y , i t p r e s e n t s i t s e l f as granulomatous, nodular abscesses i n one o r more of s e v e r a l organs, which may  i n c l u d e the lungs,- kidneys,  spleen, lymph nodes o r j o i n t s .  liver,  In the lungs, the gross  appearance i s s i m i l a r to disseminated, f a r advanced t u b e r culosis.  In t h i s study, l e s i o n s were found  spleen, lungs, and twice i n b r a i n In induced ( F i g . 2 and 13)  and  i n the l i v e r , infections  i n the mesenteric lymph nodes ( F i g . 8 ) ,  or the kidney c o r t e x ( F i g . 9 ) f o l l o w i n g i n t r a p e r i t o n e a l o r intravenous i n j e c t i o n r e s p e c t i v e l y . (63).  Only one author,  Polak  has ever r e p o r t e d s u c c e s s f u l d u p l i c a t i o n of the spon-  taneous or " n a t u r a l " form of the d i s e a s e f o l l o w i n g d i r e c t a d m i n i s t r a t i o n of v i r u l e n t organisms, Antapol  (3)  was  the f i r s t  i n t h i s case per os.  to r e p o r t the  apparent  i n d u c t i o n of c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s i n mice. The d i s e a s e f o l l o w e d p r e v i o u s treatment of l a b o r a t o r y mice with cortisone.  He noted p a r t i c u l a r l y t h a t although  ab-  scesses were found i n the l i v e r , kidney or spleen, they were never found a t the s i t e of the c o r t i s o n e i n j e c t i o n ,  77 c o n s i s t e n t w i t h a c t i v a t i o n of l a t e n c y r a t h e r than i n t r o d u c t i o n of organisms from the o u t s i d e through the punctured skin. (72),  The d i s e a s e  has s i n c e "been induced by X - i r r a d i a t i o n  lymphocytic c h o r i o m e n i n g i t i s  virus (54),  (7), e c t r o m e l i a v i r u s ( 4 8 ) ,  deficiency rickettsial  i n f e c t i o n (43),  nutritional  salmonellosis  and i n the present  (80),  study, i n -  f e c t i o n o f mice by RLV (1*5). LeMaistre  and Tompsett (49) were the f i r s t to r e p o r t  the occurrence o f the same d i s e a s e pseudotuberculosis  i n rats.  was observed i n 4 l o f 50 a l b i n o r a t s  following cortisone administration. sequently  Corynebacterial  C. k u t s c h e r i was sub-  i s o l a t e d from l e s i o n s , y e t they r e p o r t e d  that  this  organism was not demonstrably a p a r t o f the u s u a l b a c t e r i a l f l o r a o f t h e host. Pierce-Chase et a l (62) r e p o r t e d ferences  significant  dif-  i n s u s c e p t i b i l i t y of d i f f e r e n t s t r a i n s of mice to  i n j e c t i o n o f the v i r u l e n t organism.  Corynebacterial  pseudo-  t u b e r c u l o s i s sometimes developed i n r e s i s t a n t mice f o l l o w i n g c o r t i s o n e i n j e c t i o n , but never i n s u s c e p t i b l e mice. concluded t h a t s t r a i n s of mice r e s i s t a n t to are  They  pseudotuberculosis  showing r e s i s t a n c e to s u p e r i n f e c t i o n , t h a t i s , the  e n t i r e s t r a i n s o r colonies are l a t e n t l y i n f e c t e d .  T h i s con-  c l u s i o n has been supported by t h i s study, WSW mice e v i d e n t l y being r e s i s t a n t to i n j e c t i o n of C. k u t s c h e r l organisms because of a uniform p r o t e c t i o n a s s o c i a t e d w i t h l a t e n t i n f e c tion.  Comparably, WSW mice developed  pseudotuberculosis  78 f o l l o w i n g c o r t i s o n e a d m i n i s t r a t i o n or i n j e c t i o n of  RLV,  but A/J mice or CF^(SPF) mice, which l a c k r e s i s t a n c e to i  i n j e c t i o n of C. k u t s c h e r l . d i d not Pierce-Chase et a l ( 6 2 )  (Table I ) .  were u n s u c c e s s f u l  in a l l  attempts to i s o l a t e the c a u s a t i v e agent from the s t r a i n s of mice.  Fauve et a l (3*0  then hypothesized t h a t i n mice w i t h  l a t e n t i n f e c t i o n s , v i r u l e n t C. k u t s c h e r i could not  be  i s o l a t e d because the organisms e x i s t e d i n a transformed therefore unrecognizable state. p o s i t i v e , chaining,  They proposed t h a t a Gram  c o c c o i d organism ( F i g . 6 ) ,  g e n i c on i n j e c t i o n i n t o mice and other r e s p e c t s  (Table I I I ) was  and  non-patho-  q u i t e d i f f e r e n t i n many  the v a r i a n t form i n q u e s t i o n .  ( T h i s i s C. k u t s c h e r i ATCC 1 5 6 7 8 ) .  T h e i r arguments were  based mainly on c o n s i s t e n t i s o l a t i o n of the l a t t e r organism from presumed l a t e n t l y i n f e c t e d mice, and establishment  of l a t e n t c o r y n e b a c t e r i a l  the apparent pseudotuberculosis  (with r e s i s t a n c e to i n f e c t i o n by v i r u l e n t C. k u t s c h e r l ) i n mice i n j e c t e d w i t h the non-pathogenic organism.  They a l s o  noted a f a i r degree of immunological c r o s s r e a c t i o n . I n one  of the experiments done i n t h i s study, i t was  noted t h a t both organisms have l a r g e amounts of i n the c e l l w a l l  ( F i g . 7 and  Table I I I ) .  galactose  T h i s could r e s u l t  i n a s i m i l a r i t y of p a r t of the c e l l w a l l s t r u c t u r e r a t i n g galactose  and  c r o s s r e a c t i o n , and  account f o r a degree of  incorpo-  immunological  perhaps even measurable c r o s s r e s i s t a n c e ,  even i f the organisms were otherwise u n r e l a t e d .  I t could  79  not,  however, e x p l a i n away establishment Fauve et a l (3^) a l s o r e p o r t e d  of  latency.  that an S  r  (strepto-  mycin r e s i s t a n c e ) marker on a mutation of the proposed v a r i a n t s t r a i n gave r i s e to an S  r  marker on pathogenic  C. k u t s c h e r i i s o l a t e d from mice p r e v i o u s l y i n j e c t e d w i t h the former organism and  subsequently t r e a t e d with c o r t i s o n e .  I t could be argued, however, that the c o n d i t i o n s of  that  experiment simultaneously  re-  s e l e c t e d f o r streptomycin  s i s t a n t pathogenic G. k u t s c h e r l mutants q u i t e apart the occurrence of any other.  transformation  of one  from  s t r a i n to  the  Evidence to support the v a r i a n t h y p o t h e s i s was  found i n t h i s  not  study.  Caren and  Rosenberg (18)  showed that a mouse s t r a i n  w i t h hemolytic complement f a r e d s l i g h t l y b e t t e r i n r e s i s t a n c e to C. k u t s c h e r i i n j e c t i o n than a s t r a i n which d i d not. sera of most s t r a i n s of l a b o r a t o r y mice l a c k one  of  sequence complement components r e q u i r e d to complete of antibody s e n s i t i z e d e r y t h r o c y t e s ) . were i n the r e s i s t a n t category and latently, infected.  Nevertheless,  (The  the lysis  Both s t r a i n s , however,  t h e r e f o r e presumably both the experiment demonstrates  t h a t g e n e t i c f a c t o r s can i n f l u e n c e r e s i s t a n c e to t h i s o r ganism.  But  supposing t h a t a l l mice are c a r r i e r s of  organism i n the l a t e n t s t a t e , and  t h a t one  this  or more unknown  g e n e t i c f a c t o r s then determine which mice are s u s c e p t i b l e to s u p e r - i n f e c t i o n and the problem of why  which are not, one c o r t i s o n e or other  is still  faced with  immunosuppressive  80 agents would a c t i v a t e the d i s e a s e i n the most r e s i s t a n t supposedly  immunologically  complete mouse.  One  would  and  then  have to propose t h a t the most r e s i s t a n t mouse depended most h i g h l y on a f a c t o r s e n s i t i v e to c o r t i s o n e , w h i l e the  sensi-  t i v e mouse r e l i e d on a l e s s e f f i c i e n t f a c t o r which was  not.  I t seems u n l i k e l y however, t h a t i n a l l the d i v e r s e s i t u a t i o n s i n which l a t e n c y i s a c t i v a t e d , a l l the agents r e s p o n s i b l e would u n i f o r m l y behave the same way  i n any g i v e n s t r a i n of  mice, g i v e n t h a t a l l s t r a i n s were u n i f o r m l y l a t e n t l y  infected.  I s o l a t i o n of v i r u l e n t C. k u t s c h e r l from l a t e n t l y i n f e c t e d mice has been attempted i n many l a b o r a t o r i e s , always w i t h l i m i t e d or d o u b t f u l success. numbers of mice and percentage,  B i c k s (7) examined l a r g e  i s o l a t e d C. k u t s c h e r i from a very small  u n l e s s the mice examined were f i r s t  a "predisposing factor".  exposed to  I n the many l a b o r a t o r i e s i n which  the d i s e a s e has been s t u d i e d , the p a t t e r n has always been the s a m e — a spontaneous outbreak  of c o r y n e b a c t e r i a l pseudo-  t u b e r c u l o s i s i n the course of an u n r e l a t e d experiment, the i d e n t i f i c a t i o n of C. k u t s c h e r l as the e t l o l o g i c agent, where attempts have been made, extremely  limited  and  success i n  i s o l a t i o n of the agent from c o n t r o l or a p p a r e n t l y h e a l t h y mice.  The  few  i s o l a t i o n s from a p p a r e n t l y h e a l t h y mice i n  t h i s l a b o r a t o r y as w e l l as i n o t h e r s may  w e l l have been  from animals p r e d i s p o s e d or induced i n some unknown  way.  81 Immunodepresslon.  Observations made i n t h i s  study  v e r i f y the r e p o r t s of o t h e r s ( 3 , 3 4 , 4 9 ) t h a t c o r t i s o n e a d m i n i s t e r e d i n e x c e s s i v e doses i s an e f f e c t i v e i n d u c e r o f c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s i n l a t e n t l y i n f e c t e d mice and r a t s .  Some o f the e f f e c t s o f such c o r t i s o n e i n j e c t i o n  i n mice w i l l be c o n s i d e r e d i n t h i s s e c t i o n ; e s p e c i a l l y the e f f e c t s on the mouse Immune system.  The l a t t e r e f f e c t s  will  be compared to immunosuppressive e f f e c t s which have been r e p o r t e d i n murine Antopol  leukemias.  ( 3 ) i n j e c t e d 9 d a i l y doses of 2 . 5 mg o f c o r -  t i s o n e i n t o mice and some of the e f f e c t s noted were weight l o s s and decrease  i n f a t , hypertrophy  of the adrenal cortex,  atrophy o f t h e thymus and spleen, a decrease  i n lymphocytes  and an i n c r e a s e i n polymorphonuclear l e u k o c y t e s . (69)  Robinson  notes as common knowledge t h a t h i g h dose l e v e l s cause  a n e g a t i v e n i t r o g e n balance and i n f l u e n c e the f u n c t i o n a l c a p a c i t y o f many organs.  Antopol r e p o r t e d a c t i v a t i o n o f  c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s i n 8 o f 36 mice, Robinson noted  i n a d d i t i o n a lowered  r e s i s t a n c e t o acute  by K. pneumoniae, Sa. s c h o t t m u e l l e r l , Staph,  infections  aureus,  Strep,  pyogenes. P. v u l g a r i s and Ps. aeruginosa. While lowered  r e s i s t a n c e to i n f e c t i o n a p p a r e n t l y i n -  d i c a t e s impaired immune f u n c t i o n , i t i s necessary to c o n s i d e r proposed mechanisms w i t h c a u t i o n , s i n c e t h e e f f e c t s of such r e l a t i v e l y huge doses a r e q u i t e complex.  R i s c h e l ( 3 5 ) et  a l r e p o r t e d no i n c r e a s e i n d e c l i n e of e x i s t i n g antibody  titer  82 f o l l o w i n g i n j e c t i o n of c o r t i s o n e i n t o r a b b i t s , and concluded that e x i s t i n g serum p r o t e i n s were t h e r e f o r e not being broken down.  However, Bjorneboe  et a l (9)  and F i s c h e l et a l (35)  r e p o r t e d i n h i b i t i o n of antibody s y n t h e s i s by c o r t i s o n e . F i s c h e l notes that d e b i l i t y was a c t u a l l y observed  ( l o s s of weight,  morbid s t a t e )  among some of the b e t t e r antibody p r o -  ducers, i n d i c a t i n g t h a t the e f f e c t s o f c o r t i s o n e on antibody p r o d u c t i o n were not due to d e b i l i t y .  Elliott  r e p o r t e d t h a t c o r t i s o n e a c e t a t e depressed  et a l (29)  both 19S and ?S  haemolysin when a d m i n i s t e r e d p r i o r t o a n t i g e n , but depressed only 7S i f a d m i n i s t e r e d afterward.  T h i s would i n d i c a t e t h a t  c e l l s which began to produce antibody  (the e a r l y 19S r e -  spons) b e f o r e c o r t i s o n e i n j e c t i o n , continued to do so i n the presence  o f c o r t i s o n e , although an e x t e n s i o n o f the response  was blocked.  T h i s i s a l s o evidence t h a t c o r t i s o n e i s exer-  t i n g one o f i t s e f f e c t s d i r e c t l y on the plasma c e l l Kit  and B a r r o n (46)  i n 1953 r e p o r t e d an e f f e c t of a d r e n a l  c o r t i c a l hormones on the r e s p i r a t i o n of lymphatic They found d e f i n i t e i n h i b i t i o n of anaerobic T h i s suggests t h a t c o r t i s o n e may have a the lymphoid  series.  cells.  glycolysis.  d i r e c t e f f e c t on  cell.  A number of workers s i n c e 1966 have r e p o r t e d immunod e p r e s s i v e e f f e c t s o f murine leukemia v i r u s e s . Morton (74)  S i e g e l and  showed a d i r e c t r e l a t i o n between the degree o f  leukemogenesis o f mice with Rauscher d i s e a s e and the primary immune response  to sheep e r y t h r o c y t e s .  They found no  83 d e p r e s s i o n of the secondary response and no i n c r e a s e i n the r a t e of d e c l i n e of e x i s t i n g antibody authors  levels.  ( 7 5 ) a l s o r e p o r t e d depressed antibody  The same response to  bovine serum albumin when RLV was i n j e c t e d 10 days l a t e r . The  i n h i b i t i n g e f f e c t on the i n c r e a s i n g antibody  t i t e r of  the RLV i s immediate, showing t h a t the immunodepressive e f f e c t occurs The present  i n the e r y t h r o b l a s t i c stage of the d i s e a s e .  study has shown t h a t a c t i v a t i o n o f l a t e n c y must  a l s o b e g i n i n the e a r l y stages o f the Rauscher d i s e a s e . These r e s u l t s suggest a c o m p e t i t i o n between RLV and the BSA a n t i g e n f o r a stem c e l l t h a t has immunoproliferative t i a l s as w e l l as being the  poten-  s u b j e c t to n e o p l a s t i c d i r e c t i o n by  virus. Cremer et a l ( 2 1 ) r e p o r t e d immunodepression of hemag-  g l u t i n i n s i n response to i n j e c t i o n of sheep e r y t h r o c y t e s i n t o r a t s i n f e c t e d with Moloney v i r u s .  Salaman and Wedder-  burn ( 7 1 ) r e p o r t e d t h a t F r i e n d v i r u s depressed both the primary and the secondary hemagglutinin erythrocytes.  response to sheet  They r e p o r t e d the e f f e c t o c c u r r e d where v i r u s  was i n j e c t e d from 1 to 35 days before a n t i g e n was  administered.  ( F r i e n d v i r u s i n f e c t i o n l a c k s the i n i t i a l e r y t h r o b l a s t i c r e a c t i o n c h a r a c t e r i s t i c o f t h e Rauscher d i s e a s e ) . and  Schaeffer  ( 5 3 ) a l s o r e p o r t e d best i n h i b i t o r y  Millian results;  when F r i e n d v i r u s was i n j e c t e d i n t o mice 1 - 3 weeks before an a n t i g e n i c stimulus. depressant  Rauscher v i r u s a l s o had an immuno-  e f f e c t when i n j e c t e d e a r l y .  Influenza vaccine,  84 Coxsakie v i r u s , DPT and d i p h t h e r i a t o x o i d were used as a n t i gens.  Ceglowski and Friedman (20) used the haemolytic  plaque method t o determine whether mice i n f e c t e d w i t h e i t h e r the F r i e n d o r Rauscher leukemia v i r u s showed any i n c r e a s e i n antibody forming c e l l s f o l l o w i n g i n j e c t i o n of sheep erythrocytes. of  They found t h a t no Increase i n t h e numbers  antibody forming c e l l s o c c u r s i n the v i r u s i n f e c t e d mice,  i n d i c a t i n g that the v i r u s i s e l i m i n a t i n g o r causing the r e d i r e c t i o n of plasma c e l l p r e c u r s o r s . I d e n t i f i c a t i o n o f the RLV t a r g e t c e l l  (or c e l l s )  whose i n f e c t i o n may l e a d to a c t i v a t i o n of l a t e n t  coryne-  b a c t e r i a l p s e u d o t u b e r c u l o s i s would a l s o be o f v a l u e i n exp l a i n i n g the course of RLV i n f e c t i o n , and would c o n t r i b u t e to  our understanding of the immune response.  experiments  I n one of the  considered f o r t h i s study an attempt was to be  made to c o r r e l a t e the extent of r e t l c u l o c y t o s i s i n e a r l y Rauscher d i s e a s e to the antibody response to C. k u t s c h e r l vaccine. to  I f the antibody t i t e r was i n v e r s e l y p r o p o r t i o n a l  t h e r e t i c u l o c y t e percentage a t the time t h e a n t i g e n was  administered, the r e s u l t s would be c o n s i s t e n t w i t h the theory o f S i e g e l and Morton.  I n the p r e l i m i n a r y t r i a l ,  ever, r e t l c u l o c y t o s i s appeared istic  i n i n d i v i d u a l mice (Table  how-  to be an u n s t a b l e c h a r a c t e r I I ) and the antibody r e -  sponse was d o u b t f u l i n almost a l l the t e s t mice i f there was any r e t l c u l o c y t o s i s a t a l l .  One exception, a mouse which  developed p s e u d o t u b e r c u l o s i s , had a C. k u t s c h e r l antibody t i t e r of  320.  85 The  complexity  d i s e a s e and  i n the development of the Rauscher  i t s e f f e c t i n the a c t i v a t i o n of l a t e n c y can  be  more r e a d i l y e x p l a i n e d i f i t be allowed t h a t the t a r g e t c e l l of  the v i r u s i s a p l u r i p o t e n t i a l  stem c e l l .  C e r t a i n l y the  c e l l s i n v o l v e d i n Rauscher d i s e a s e are d e r i v e d from stem c e l l s t h a t are m o r p h o l o g i c a l l y a l i k e .  Popp ( 6 4 ) demonstrated  that nucleated p e r i p h e r a l blood c e l l s are capable of r e p l a c i n g the e r y t h r o c y t e p o p u l a t i o n i n X - i r r a d i a t e d c o i s o g e n i c mice, p r o v i n g that c i r c u l a t i n g l y m p h o c y t e - l i k e d i f f e r e n t i a t e into e r y t h r o p o i e t i c c e l l s . c e l l s of the germinal  The  cells  can  proliferating  c e n t e r s i n r a t s p l e e n have been shown  by F l i e d n e r et a l ( 3 7 ) to have no g e n e t i c r e l a t i o n to the c e l l s of the margin, c o n s i s t e n t w i t h the theory t h a t they too c o u l d d e r i v e from a migrant lymphoid c e l l .  Yoffey ( 8 1 )  has p o s t u l a t e d t h a t p r i m i t i v e r e t i c u l a r c e l l s may  multiply  to a " t e r m i n a l " stage, a small lymphocyte which i s a p l u r i p o t e n t i a l s o r t of " c e l l u l a r spore".  This c e l l  circulates,  s e t t l e s where needed and d i f f e r e n t i a t e s a c c o r d i n g to r e g i o n a l stimuli.  Y o f f e y has q u a l i f i e d h i s hypothesis,  however, by  s t a t i n g that i f there are p l u r i p o t e n t i a l small lymphocytes, there are a l s o " c o n d i t i o n e d " ones, whose development, i n the c e l l  c o n d i t i o n e d to become a plasma c e l l , would depend  on i t s eventual chance p o s i t i o n i n g among the proper c e l l s i n lymphoid t i s s u e and r e c e i v i n g the proper The  say,  support  stimulus.  consensus at the p r e s e n t time supports a heterogenous,  even though m o r p h o l o g i c a l l y s i m i l a r , lymphocyte p o p u l a t i o n  86 (30,  39).  S i e g e l and Morton  (75)  would have the RLV In-  f e c t i o n a c t a t o r b e f o r e the c o n d i t i o n i n g stage, so t h a t no such c e l l s of complete plasmocyte p o t e n t i a l a r e produced. Instead, an excess of c e l l s having e r y t h r o b l a s t i c p o t e n t i a l (or o t h e r ) a r e produced.  T h i s could l e a d , as observed, to  p r o l i f e r a t i o n of e r y t h r o b l a s t i c c e l l s and an absence of  new  plasmocytes, r e s u l t i n g i n r e t l c u l o c y t o s i s and the i n a b i l i t y of the d i s e a s e d mouse to produce new antibody. Phagocytosis In v i t r o .  The f i r s t  study r e p o r t e d o f  p h a g o c y t o s i s o f C. k u t s c h e r l i n v i t r o was by Fauve and P i e r c e Chase  (33).  A counted suspension of mouse p e r i t o n e a l  cells  i n h e p a r i n i z e d Hanks' s o l u t i o n was mixed w i t h a suspension of b a c t e r i a l c e l l s and incubated.  When samples were removed  at i n t e r v a l s , d i l u t e d s e r i a l l y and p l a t e d onto n u t r i e n t agar, i t was r e p o r t e d that fewer c o l o n i e s grew when the source of the p e r i t o n e a l exudate was a mouse r e s i s t a n t to C. k u t s c h e r i i n f e c t i o n , t h a t i s , a l a t e n t l y i n f e c t e d mouse.  T h i s method  d i d d i f f e r e n t i a t e between b a c t e r i c i d a l a c t i v i t y of the exudate and simple p h a g o c y t o s i s , because most phagocytes would remain i n t a c t and each phagocyte c o n t a i n i n g any number of v i a b l e b a c t e r i a would r e s u l t i n the growth o f one colony. L a t e r , Fauve  (3D  which d i f f e r e d  conducted a second s e r i e s of experiments  i n two important r e s p e c t s .  Only the c e l l s  which adhered to the g l a s s w a l l s o f c u l t u r e tubes  (macro-  phage) were used i n the study, and the c e l l s were ruptured  87 by  s o n i c a t i o n b e f o r e p l a t i n g as before.  These experiments  showed t h a t whereas the r a t e of p h a g o c y t o s i s was s i m i l a r regardless  of the source o f the macrophage (C. k u t s c h e r i  r e s i s t a n t o r s u s c e p t i b l e mice), death of the b a c t e r i a l c e l l s evidently-occurred  only when the macrophages were from C.  k u t s c h e r l r e s i s t a n t and presumed l a t e n t l y i n f e c t e d mice. Fauve et a l ( 3 2 ) r e p o r t e d C. k u t s c h e r l  in a still  l a t e r experiment t h a t  i s a c t u a l l y a b l e to m u l t i p l y  of s u s c e p t i b l e mice.  i n s i d e macrophages  I f these s u s c e p t i b l e mice were f i r s t  immunized w i t h L. monocytogenes, m u l t i p l i c a t i o n occurred a t a slower r a t e .  In the f o r e g o i n g  where macrophages were c u l t u r e d , supplemented w i t h c a l f serum.  sequence of experiments, the c u l t u r e medium was  Whether C. k u t s c h e r l  were  k i l l e d o r a t what r a t e they m u l t i p l i e d w i t h i n the macrophages depended on f a c t o r s a s s o c i a t e d w i t h the c e l l s r a t h e r opsonins i n the medium. ported  Nelstrop  than  et a l ( 5 8 ) r e c e n t l y r e -  t h a t washed, c u l t u r e d macrophages from r a b b i t s p r e -  v i o u s l y immunized w i t h T-j_ bacteriophage were more e f f i c i e n t i n the p h a g o c y t o s i s of  i n v i t r o than were macrophages  from non-immunized c o n t r o l r a b b i t s .  In t h i s case as w e l l ,  the macrophages were p r e v i o u s l y washed and no  detectable  n e u t r a l i z i n g antibody was present i n the c u l t u r e medium. In any case, any o p s o n i z i n g  f a c t o r s present i n the medium  of the c u l t u r e s of immune c e l l s would a l s o be present i n the c o n t r o l s .  I t was t h e r e f o r e a g a i n concluded that the  phagocytes were immune o r p r e - c o n d i t i o n e d  i n some way.  88 It  i s l o g i c a l that an i n c r e a s e d r a t e of i n g e s t i o n by  phagocytic  c e l l s could be accounted f o r by  " c e l l bound" a n t i -  body, g l o b u l i n contents of the donor serum which become attached  to the p h a g o c y t i c  cell  i n such a way  t a t e p h a g o c y t o s i s apart from the o p s o n i z i n g bodies i n the medium i t s e l f .  as to  facili-  e f f e c t of  Work by Old et a l (60)  antiseems  to i n d i c a t e , however, t h a t such antibody can be washed o f f . In any  case, i t seems d o u b t f u l  would be  that c e l l - b o u n d  s u f f i c i e n t to account f o r Increased  of the i n g e s t e d  inactivation  bacterial cell.  Maxwell and  Marcus (52)  showed t h a t p e r i t o n e a l  guinea p i g a l v e o l a r macrophages harvested v i o u s l y Immunized w i t h BCG M.  antibody  from animals p r e -  were b e t t e r a b l e to i n a c t i v a t e  t u b e r c u l o s i s l n t r a c e l l u l a r l y than were c o n t r o l macrophages  from non-immune animals.  They concluded that phagocytes  from BCG-immunized animals had  increased  cytopeptic  when compared to phagocytes from normal animals. al  and  (41) p r e v i o u s l y r e p o r t e d  t h a t normal and  activity  Heise et  immune a l v e o l a r  and p e r i t o n e a l phagocytes d i f f e r i n lysozyme content. E i t h e r o r both c i r c u l a t i n g and still  be  cell  bound antibody  could  i n v o l v e d i n i n g e s t i o n i f not at least p a r t l y i n  subsequent d e s t r u c t i o n of i n g e s t e d b a c t e r i a . The  above r e p o r t s are c o n s i s t e n t w i t h the hypothesis  t h a t s p e c i f i c , a n t i b o d y i n the medium (or bound to cells) facilitates  phagocytic  (but i s not e s s e n t i a l to) phagocytosis by  p e r i t o n e a l macrophage i n v i t r o ; but  subsequent d e s t r u c t i o n  89  of the  ingested, b a c t e r i a depends on f a c t o r s inherent  phagocytic c e l l  i t s e l f , which are s p e c i f i c to the  v a t i o n of c e r t a i n b a c t e r i a l s p e c i e s q u i t e apart presence of any  i n the  inacti-  from  the  s p e c i f i c antibody.  Taking the f o r e g o i n g  into consideration,  in vitro  p h a g o c y t o s i s experiments used i n t h i s study were designed to show whether the r a t e of i n g e s t i o n (phagocytic  index) of  C. k u t s c h e r i by pooled, washed p e r i t o n e a l macrophage taken from a g i v e n  s t r a i n of mice would vary a c c o r d i n g  to  the  d i f f e r e n t s t a t e of the mice from which serum supplement f o r the c u l t u r e medium was  taken.  Thus, w i t h the phagocytes as  a constant f a c t o r , w i l l macrophage from A/J mice (a C. kutscheri  s u s c e p t i b l e mouse) i n g e s t G. k u t s c h e r l a t a  greater  r a t e i f they are exposed to serum from C. k u t s c h e r i r e s i s t a n t mice?  Conversely, i f the macrophages are taken from r e s i s -  t a n t mice, w i l l the p h a g o c y t i c index of c e l l s bathed i n the pooled sera of C. k u t s c h e r i r e s i s t a n t but RLV be l e s s than i f the non  leukemic?  The  serum was  i n f e c t e d mice  from r e s i s t a n t mice t h a t were  answer to these q u e s t i o n s depends on  the  presence o r absence of a humoral f a c t o r i n the s e r a of r e s i s t a n t mice able to s t i m u l a t e  p h a g o c y t o s i s but  a b l e as c i r c u l a t i n g s p e c i f i c antibody.  The  not  detect-  r e s u l t s of t h i s  experiment i n d i c a t e d t h a t such a f a c t o r i s present i n leukemic and and  non-  sometimes leukemic C. k u t s c h e r i r e s i s t a n t mice  i s absent i n s u s c e p t i b l e mice.  t h a t t h i s f a c t o r confers  I t i s by no means proved  r e s i s t a n c e , even though i t i s found  90  i n r e s i s t a n t mice.  I t would be l o g i c a l to assume t h a t i t  would c o n t r i b u t e to r e s i s t a n c e i f the p h a g o c y t i c c e l l s were a b l e to i n a c t i v a t e the i n g e s t e d b a c t e r i a .  I f not, i t has  been shown that a c t i v e phagocytosis by macrophages can a c t u a l l y be a means of spreading i n f e c t i o n throughout organism.  F a u v e s o b s e r v a t i o n (31)  t h a t C. k u t s c h e r i  1  m u l t i p l y a c t i v e l y i n s i d e p e r i t o n e a l macrophages of mice i n d i c a t e s t h a t k i l l i n g  the  sensitive  power as w e l l as p h a g o c y t i c  power i s e s s e n t i a l i n c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s , j u s t as has been shown to be the case i n t u b e r c u l o s i s . Clearance s t u d i e s . al  I t was  (5) t h a t the laws governing  r e p o r t e d by Benacerraf  et  the c l e a r a n c e of b a c t e r i a ,  from the blood of mice and o t h e r l a b o r a t o r y animals were the same as was  p r e v i o u s l y shown to apply f o r c o l l o i d a l  pensions  Below a c r i t i c a l p o i n t where a l l a v a i l a b l e  (24).  sus-  RES p h a g o c y t i c s i t e s have become s a t u r a t e d , the c o n c e n t r a t i o n of  b a c t e r i a i n the c i r c u l a t i n g blood decreases a c c o r d i n g to  an e x p o n e n t i a l f u n c t i o n of the time. K,  The  c l e a r a n c e constant,'  then depends on the c o n c e n t r a t i o n of b a c t e r i a l  cells  present and the a v i d i t y w i t h which they a r e phagocytosed. The k i n e t i c s of c l e a r a n c e under these c o n d i t i o n s can be d e s c r i b e d by the  equation: K = log C  where l o g C^, - l o g C  2  1  - log  C  2  i s the change i n the l o g of the con-  c e n t r a t i o n of c i r c u l a t i n g b a c t e r i a and T  2  - T^ i s the  interval  91  over which t h i s change i s measured. Benacerraf  e t a l (4) a l s o showed t h a t the v a l u e o f K  v a r i e s from one microorganism  to the next and t h a t i n phago-  c y t o s i s o f b a c t e r i a , i n c r e a s e s i n K depend p r i m a r i l y on the a b i l i t y o f the host to opsonize the organism. i s added w i t h the organism, K w i l l  I f antibody  i n c r e a s e up to a maximum  v a l u e which i s e v i d e n t l y dependent on p h y s i c a l o r anatomical factors.  Because of i t s l a r g e r blood supply, the l i v e r i s  the p r i n c i p a l f i l t e r f o r removing b a c t e r i a , which a r e phagocytosed by the K u p f f e r c e l l s . courses v e r y r a p i d l y through  I n the mouse, the blood the l i v e r and chances of t h e i r  random c o n t a c t w i t h the K u p f f e r phagocytes a r e reduced.  The  maximum c l e a r a n c e constant K f o r b a c t e r i a i n the mouse i s t h e r e f o r e approximately antibody  0.40 which i s independent of the  titer.  A c c o r d i n g t o B i o z z i e t a l (8) p h a g o c y t o s i s o f i n e r t particles  ( c o l l o i d a l carbon) by the c e l l s of the RES i s  independent of the c o n c e n t r a t i o n o f serum opsonins.  Inges-  t i o n o f carbon should t h e r e f o r e be a r e l i a b l e method f o r i n v e s t i g a t i n g the RES f u n c t i o n i n n o n - s p e c i f i c r e s i s t a n c e , independently  o f the c o n t r i b u t i o n of humoral f a c t o r s .  k i n e t a l (42) r e p o r t e d t h a t n o n - s p e c i f i c opsonins ingestion of inert p a r t i c l e s .  Jen-  stimulated  However, the work o f B i o z z i  and o t h e r s c o n t r a d i c t e d t h i s r e s u l t .  The d i f f e r e n c e s ob-  served could be due to RES s t i m u l a n t s o r depressants which have n o t h i n g t o do w i t h o p s o n i z a t i o n .  Thorbeck and  92 Benacerraf  (77)  reported  that clearance  germfree than i n normal animals. e f f e c t of r a t e of RES Bohme (10) r e p o r t e d  r a t e s are lower i n  With regard  p h a g o c y t o s i s on d i s e a s e  c o n f l i c t i n g evidence.  to the  net  resistance,  Carbon  clearance  r a t e s were not n e c e s s a r i l y connected w i t h the a b i l i t y resist  i n f e c t i o n by S. typhlmurlum, but pyrogens, which  stimulate  the a c t i v i t y of the r e t i c u l o e n d o t h e l i a l system,  a l s o gave s i g n i f i c a n t p r o t e c t i o n to mice a g a i n s t M. I t i s p o s s i b l e to s a t u r a t e system of any  animal by  et a l ( 2 5 )  i n j e c t i n g numbers of Organisms which  i . e . , the RES  type of p a r t i c l e w h i l e s t i l l  others. factor,  cells.  showed t h a t t h i s phenomenon, c a l l e d  Is p a r t i c l e s p e c i f i c ,  The  fortultum.  the r e t i c u l o e n d o t h e l i a l  exceed the t o t a l c a p a c i t y of the p h a g o c y t i c  one  to  Drutz  "blockade",  can be blockaded f o r  a c t i v e l y phagocytizing  r e a s o n i s not d e p l e t i o n of a s p e c i f i c humoral  s i n c e a d d i t i o n of f r e s h serum does not break the  blockade.  They b e l i e v e blockade to be a c e l l  surface  phenomenon r a t h e r than a p h y s i c a l s t u f f i n g of the RES Nelstrop  et a l (58)  have observed enhanced  when a second i n j e c t i o n of a n t i g e n i c m a t e r i a l , work T^ and  T  2  phage, was  w i t h i n 24 hrs, too detectable  given.  clearance  in their  This s t i m u l a t i o n occurs  soon to be a t t r i b u t e d to the presence of  c i r c u l a t i n g antibody.  occur when u s i n g non-antigenlc  T h i s phenomenon does not  material  In view of the f o r e g o i n g , 0.40  cells.  such as carbon.  the c l e a r a n c e  observed f o r C. k u t s c h e r l by WSW  mice not  constant  of  demonstrating  93  a d e t e c t a b l e t i t e r of c i r c u l a t i n g antibody high.  i s remarkably  T h i s might be e x p l a i n e d on the b a s i s o f a "precon-  d i t i o n i n g " phenomenon o f RES c e l l s f o r phagocytosing organisms i n the l a t e n t l y i n f e c t e d mice.-  these  On the other hand,  the h i g h constant may be due to p h y s i c a l f a c t o r s .  C.  k u t s c h e r i c e l l s have a strong tendency to a u t o - a g g l u t i n a t e . The  same f o r c e s of adhesion t h a t b r i n g the b a c t e r i a l  cells  t o g e t h e r may a l s o be b r i n g i n g clumps of b a c t e r i a i n c l o s e p r o x i m i t y t o the s u r f a c e of the phagocytic greatly f a c i l i t a t i n g  cells,  thus  clearance.  As r e p o r t e d by Dobson (24), the b i g g e r the p a r t i c l e , the f a s t e r i t s removal.  Thus, the f a c t t h a t C. k u t s c h e r i  clump so r e a d i l y would i n c r e a s e c l e a r a n c e on the b a s i s o f s i z e alone.  I t was t h e r e f o r e considered  important  experiments to minimize the clumping e f f e c t . of uniform rates.  i n these  C l u s t e r s not  s i z e would a l s o be phagocytosed a t d i f f e r e n t  Such an e f f e c t would r e a d i l y be observed by p l o t t i n g  s e v e r a l p o i n t s i n the e x p o n e n t i a l  clearance p l o t .  Hetero-  g e n i e t y of p a r t i c l e s i z e would not produce p o i n t s i n a straight line,  s i n c e i n d i v i d u a l b a c t e r i a l would continue t o  be phagocytosed a t a lower K a f t e r a l l the clumps had a l r e a d y been removed. A s i d e from a l l o t h e r c o n s i d e r a t i o n s , the c l e a r a n c e constant decreases i n a l l cases when the c o n c e n t r a t i o n o f c i r c u l a t i n g b a c t e r i a drops t o l e s s than 10$ o f the o r i g i n a l l y i n j e c t e d dose (5).  T h i s phenomenon has not been  completely  94 e x p l a i n e d , but may  be due  to subsequent r e l e a s e of p r e -  v i o u s l y bound o r phagocytosed c e l l s .  Thus, p o i n t s on  the  e x p o n e n t i a l curve which are p l o t t e d a t the lower v a l u e s of c i r c u l a t i n g b a c t e r i a present a r e ignored i n c a l c u l a t i n g c l e a r a n c e constants.  With C. k u t s c h e r l , the most depend-  a b l e r e s u l t s are o b t a i n e d w i t h i n f o u r minutes of the  time  the intravenous i n j e c t i o n of l a b e l e d organisms i s made. Summary and  conclusions.  The problem under study,  F a c t o r s C o n t r i b u t i n g to the Maintenance and A c t i v a t i o n of C o r y n e b a c t e r i a l P s e u d o t u b e r c u l o s i s i n the Laboratory Mouse, was  motivated by two  related observations:  1. A c t i v e c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s , the c a u s a t i v e agent of which i s the bact e r i u m Cornebacterium i n WSW  k u t s c h e r l , appeared  mice which had been i n j e c t e d w i t h  the Rauscher murine leukemia v i r u s , but never found  i n RLV  2. Large numbers of WSW  f r e e WSW  was  controls.  mice, i f not a l l the  mice i n the colony, have an inapparent  or  masked C. k u t s c h e r l i n f e c t i o n , t h a t i s , they have l a t e n t c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s . During the f i r s t phase of t h i s study, u s i n g 603 mice were designed the above o b s e r v a t i o n s and  experiments  to t e s t the r e p r o d u c i b i l i t y of  to o b t a i n some q u a n t i t a t i v e data.  These data appear i n Tables I, V, and  VII.  Table I shows  95 t h a t of the mice t h a t were i n j e c t e d with RLV quently 16.5$ and  succumbed to the Rauscher d i s e a s e ,  had 5% had  a c t i v e pseudotuberculosis. nor  approximately  mice i s c r i t i c a l .  Table I a l s o shows  sex i s an important f a c t o r i n i n d u c t i o n  of the c o r y n e b a c t e r i a l d i s e a s e , but  not  subse-  c u l t i v a t a b l e C. k u t s c h e r i i n t h e i r i n t e r n a l organs  t h a t n e i t h e r age  but  and  that the  Thus, a c t i v a t i o n may  source of  occur i n WSW  the  mice,  i n the A/J or CF (SPF) animals. 1  T a b l e s V and considered  together,  VII have a s p e c i a l s i g n i f i c a n c e when because they show that WSW  are r e s i s t a n t to i n j e c t i o n of i o 5 v i r u l e n t C.  mice, which kutscherl  organisms, are the mice which develop c o r y n e b a c t e r i a l pseudot u b e r c u l o s i s f o l l o w i n g i n j e c t i o n of c o r t i s o n e , and t h a t A/J and of 10^  conversely,  CF]_(SPF) mice, which are s u s c e p t i b l e to i n j e c t i o n  organisms, never develop c o r y n e b a c t e r i a l pseudotuber-  c u l o s i s i n response to e i t h e r c o r t i s o n e or RLV  administration.  T h i s i s c o n s i s t e n t w i t h the r e p o r t of Fauve et a l (3*0  that  e n t i r e c o l o n i e s of mice tend to become l a t e n t l y i n f e c t e d and that r e s i s t a n c e to exogenous i n f e c t i o n accompanies l a t e n c y . In c o n s i d e r a t i o n of the f o r e g o i n g , t h a t the e n t i r e WSW  colony  i t i s considered  i s l a t e n t l y i n f e c t e d and  a c t i v e corynebacterial pseudotuberculosis  probable that  which developed  i n these mice a f t e r Rauscher v i r u s i n f e c t i o n d i d so as  an  i n d i r e c t r e s u l t of the development of the Rauscher d i s e a s e . Once the l a t e n t s t a t e of c o r y n e b a c t e r i a l pseudotuberc u l o s i s had  been confirmed f o r WSW  mice, and  i t s activation  96 by RLV had been demonstrated as r e p r o d u c i b l e , f u r t h e r s t u d i e s were i n i t i a t e d to determine some of the f a c t o r s which c o n t r i b u t e d t o the maintenance and a c t i v a t i o n of the latent state.  The general approach to the problem was t o  a d m i n i s t e r the i n d u c i n g agent  (RLV) to l a t e n t l y  infected  mice and to observe any changes which took p l a c e i n the host defense systems d u r i n g the normal a c t i v a t i o n p e r i o d , with a view to r e l a t i n g these changes to decreased  r e s i s t a n c e to  the m u l t i p l i c a t i o n and d i s s e m i n a t i o n o f C. k u t s c h e r i .  Clues  were a l s o sought i n the p h y s i c a l and b i o c h e m i c a l p r o p e r t i e s of v i r u l e n t C. k u t s c h e r l and a proposed a v i r u l e n t v a r i a n t (C. k u t s c h e r l ATCC 15678); and i n comparisons of the l a t e n t l y I n f e c t e d and C. k u t s c h e r i f r e e mouse s t r a i n s w i t h regard to d i f f e r e n c e s i n t h e i r r e s p e c t i v e responses to immunological s t i m u l i w i t h C. k u t s c h e r i v a c c i n e , o r intravenous  administra-  t i o n of v a r i o u s numbers of v i r u l e n t organisms. The f o l l o w i n g changes In the l a t e n t l y i n f e c t e d mouse were observed  w i t h i n 14-21 days o f a d m i n i s t r a t i o n of  s u f f i c i e n t RLV to i n f e c t the animal. 1. An i n c r e a s e i n the numbers of r e t i c u l o c y t e s (immature e r y t h r o c y t e s ) i n the c i r c u l a t i n g blood  (Table I I ) .  2. The disappearance  of unknown,  f a c t o r s which were present f e c t e d WSW  WSW  n a t u r a l serum  i n non-RLV i n -  mice and which were a b l e to  s t i m u l a t e i n v i t r o p h a g o c y t o s i s of  97  C. k u t s c h e r l by mouse p e r i t o n e a l macrophage (Table V I I I ) . 3. A d e f i n i t e i n h i b i t i o n of the a b i l i t y to respond immunologically to the a d m i n i s t r a t i o n of heat k i l l e d  C. k u t s c h e r l v a c c i n e  4,. A decrease i n the r a t e of c l e a r a n c e  (Table I X ) . o f P^  2  l a b e l e d C. k u t s c h e r i from the c i r c u l a t i n g blood The  (Fig. 1 7 ) .  f i r s t observation  i s more d i f f i c u l t to b r i n g  d i r e c t l y to bear on the problem a t hand because the e r y t h r o cyte has no obvious connection I t seems l o g i c a l , more general  w i t h immunological defense.  however, that r e t l c u l o c y t o s i s r e f l e c t s a  c o n d i t i o n t h a t c o u l d have profound e f f e c t on  a c t i v a t i o n of l a t e n t i n f e c t i o n .  That i s , d i s e a s e  and d i s -  o r g a n i z a t i o n which begins i n r e t i c u l a r t i s s u e and very  soon  a f f e c t s the replacement and f u n c t i o n o f most, i f not a l l c e l l s a s s o c i a t e d w i t h immunological defense.  This  includes  the plasma c e l l and the phagocytosing c e l l s of the RES. In t h i s connection  the gross appearance and d i s t r i -  bution of the l e s i o n s of corynebacterial  pseudotuberculosis  which was induced by RLV o r c o r t i s o n e was never d u p l i c a t e d by a d m i n i s t r a t i o n o f v i r u l e n t organisms (see Cortisone s t r a t i o n i n Results  section).  Thus, intravenous  of 1 0 ? v i r u l e n t C. k u t s c h e r l i n t o WSW  injection  mice e v e n t u a l l y r e s u l t s  i n m u l t i p l e abscesses i n the r e n a l cortex, and  admini-  although Caren  Rosenberg have shown t h a t 70% o f the organisms a r e  98 initially  c l e a r e d by the l i v e r , and o n l y 1% axe  c l e a r e d by  the kidneys.  Since l e s i o n s f r e q u e n t l y appear i n the  liver  f o l l o w i n g RLV  o r c o r t i s o n e induced d i s e a s e , i t seems l o g i c a l  to  assume t h a t a change i n the a b i l i t y of the K u p f f e r  of  the l i v e r to d e s t r o y C. k u t s c h e r i must have occurred. The  changes noted  i n the next three  cells  observations  could be e x p l a i n e d s o l e l y on the b a s i s of d i s e a s e to the plasma c e l l ,  and f u r t h e r work xvould be needed to determine  whether the c y t o l y t i c powers of phagocytosing  c e l l s were  d i r e c t l y a f f e c t e d i n e a r l y Rauscher d i s e a s e , apart from the c o n t r i b u t i o n of s p e c i f i c antibody.  I t should be  noted  a g a i n a t t h i s p o i n t t h a t p r e v i o u s r e p o r t s show p r e - e x i s t i n g l e v e l s of antibody  are not d e p l e t e d d u r i n g  immunodepression.  With regard to other approaches to the problem i n t h i s study,  taken  attempts to t r a n s f e r l a t e n c y by i n j e c t i o n of  organ homogenates obtained from l a t e n t l y i n f e c t e d mice i n t o C. k u t s c h e r i f r e e mice which were simultaneously t r e a t e d w i t h c o r t i s o n e were not s u c c e s s f u l .  T h i s i s considered  i n d i c a t e t h a t i f C. k u t s c h e r i organisms are present t i s s u e i n v i r u l e n t form or otherwise,  to  i n such  t h e i r numbers are too  small to be both t r a n s f e r r e d and d e t e c t e d by t h i s method. Comparison of the immunological response to C. k u t s c h e r l v a c c i n e of l a t e n t l y  I n f e c t e d mice to C. k u t s c h e r l  f r e e mice showed an e q u a l l y h i g h response by e i t h e r (Table IX).  strain  D i f f e r e n c e s i n response to C. k u t s c h e r i a n t i g e n  are t h e r e f o r e probably  not a f a c t o r i n maintenance of  99  latency.  E l e c t r o n microscopy showed t h a t l y s i s of  virulent  C. k u t s c h e r i o c c u r s i n the polymorphonuclear l e u k o c y t e s l a t e n t l y i n f e c t e d mice ( F i g . 18C).  Fauve (32)  showed t h a t  the organism s u r v i v e s and m u l t i p l i e s i n macrophages. t h i s respect, Fig.  12  C. k u t s c h e r i resembles M.  of  In  tuberculosis.  shows i n f i l t r a t i o n of a c o r y n e b a c t e r i a l l e s i o n by  monocytic c e l l s .  I t i s probable that i n g e s t i o n of C.  c h e r l by organisms t h a t are unable to d e s t r o y butes to the d i s s e m i n a t i o n  and maintenance of  kuts-  i t , contricorynebacterial  pseudotuberculosis. S t u d i e s on the biochemical virulent  C. k u t s c h e r i and  and p h y s i c a l p r o p e r t i e s of  i t s presumed a v i r u l e n t v a r i a n t  r e v e a l e d p r o p e r t i e s which could have a b e a r i n g on a b i l i t y of the organism to p e r s i s t i n the host. of these was of s t e r i l e effect tion).  The  the l a c k of potent t o x i c p r o p e r t i e s .  first  Injection  c u l t u r e supernatants were without p e r c e p t i b l e  (see T o x i c p r o p e r t i e s i n M a t e r i a l s and Low  the  t o x i c i t y was  the comparatively  Methods sec-  demonstrated more d r a m a t i c a l l y  huge abscesses which developed i n  genously i n f e c t e d mice w i t h minimal evidence of t h a t could not be a t t r i b u t e d to pressure v i t a l organs d u r i n g  on,  by  exo-  discomfort  or growth i n  the l a t e stages of advanced  chronic  i n f e c t i o n ( F i g . 8). D i f f e r e n c e s i n the morphology, the metabolism, c e l l w a l l s t r u c t u r e and  the base composition of the  t i v e DNAs of C. k u t s c h e r l ATCC 1 5 6 7 7 and  the  respec-  i t s presumed  100 a v i r u l e n t v a r i a n t , ATCC 1 5 6 7 8 , were found to be (Table  III).  ferences  The  comparison then, has  which are not  extensive  shown major d i f -  c o n s i s t e n t w i t h the theory that  l a t t e r i s a v a r i a n t of the former.  For t h i s reason,  the  the  p o s s i b l e r o l e of the suggested v a r i a n t i n l a t e n t i n f e c t i o n was  not  i n v e s t i g a t e d f u r t h e r i n t h i s study.  The  difficulty  i n i s o l a t i n g v i r u l e n t C. k u t s c h e r l from l a t e n t l y i n f e c t e d mice does, however, support the p o s s i b i l i t y of some k i n d v a r i a t i o n occurring  i n v i v o , and  L-forms of C. k u t s c h e r i may  the p o s s i b i l i t y  that.  be present i n the t i s s u e s of  l a t e n t l y I n f e c t e d mice should be Finally,  investigated.  a theory of the mechanism of a c t i v a t i o n of  l a t e n t c o r y n e b a c t e r i a l p s e u d o t u b e r c u l o s i s by RLV i s advanced.  of  While s p e c u l a t i v e  i n part, i t i s  infection considered  c o n s i s t e n t w i t h the changes which have been observed to occur i n the RLV  i n f e c t e d WSW  of d i s e a s e .  low  The  mouse d u r i n g  the e a r l y weeks  t o x i c i t y of C. k u t s c h e r l and  the  d i f f i c u l t y experienced i n i s o l a t i n g the organism from l a t e n t l y i n f e c t e d mice a l s o give  some support to proposed  sequence of events which f o l l o w s : 1.  Latency of c o r y n e b a c t e r i a l  pseudotuberculosis  i s maintained by the r e s i d e n c e c a t i o n of C. k u t s c h e r i  and  multipli-  i n c e l l s unable to b r i n g  about i t s complete i n a c t i v a t i o n .  From time  to time as these c e l l s d i e the organisms are released  i n t o the blood stream.  The  greater  101 percentage a r e trapped by the K u p f f e r c e l l s of  the l i v e r , where they a r e d e s t r o y e d by  relatively  active l y t i c  systems.  A much  s m a l l e r percentage a r e i n g e s t e d by phagoc y t e s l a c k i n g the necessary system. toxicity,  cytolytic  Because the organism  has low  these c e l l s s u r v i v e f o r some  time, m a i n t a i n i n g the l a t e n c y . 2.  RLV invades and i s r e p l i c a t e d by monocytic c e l l s which were d e s t i n e d to d i f f e r e n t i a t e or develop i n t o c e l l s of the e r y t h r o c y t e s e r i e s , the plasma c e l l  s e r i e s , phago-  c y t i c RES c e l l s and o t h e r s .  The r e s u l t i s  d e s t r u c t i o n of many o f the immediate p r e c u r s o r s o f the l a t t e r  c e l l s , and exces-  s i v e s t i m u l a t i o n o f stem c e l l s .  Exces-  s i v e s t i m u l a t i o n and e a r l y r e l e a s e does not permit these c e l l s to develop i.e.,  normally,  e r y t h r o c y t e s have a shortened  life  span (14), de novo antibody s y n t h e s i s i s inhibited  ( 7 5 ) , and RES c e l l s presumably  do not have a f u l l  complement of a c t i v e  lysosomes (41). 3. C e l l s r e p l a c i n g , f o r example, the K u p f f e r c e l l s of the l i v e r and the a l v e o l a r macrophage of the lungs under the above  c o n d i t i o n s are not a b l e to i n a c t i v a t e h i g h l y r e s i s t a n t organisms  such as  C. k u t s c h e r i which they normally from the c i r c u l a t i n g blood and The organisms  digest.  therefore multiply  c e l l u l a r ^ and d e s t r o y the c e l l . RES  intraSurviving  c e l l s of h i g h c y t o l y t i c a c t i v i t y  g r a d u a l l y overwhelmed. confluent and  filter  liver.  are  The r e s u l t i s  nodular abscesses i n the lungs  103  C. KUTSCHERI FREE MOUSE  SUB-LETHAL C. KUTSCHERI INFECTION  V LATENTLY INFECTED MOUSE  4 10* O.K. CELLS INTRAVENOUSLY  10 MG CORTISONE INTRAPERITONEALLY  FATAL PSEUDOTUBERCULOSIS  10^ C.K. CELLS INTRAVENOUSLY  10 MG CORTISONE INTRAPERITONEALLY  NO PSEUDOTUBERCULOSIS EVIDENT  F i g . 1 9 , Hypothesized r e s i s t a n c e to s u p e r i n f e c t i o n , and presumed a c t i v a t i o n of l a t e n t c o r y n e b a c t e r i a l pseudot u b e r c u l o s i s , w i t h regard to i n f e c t i o n o f mice by C. k u t s c h e r i  104  NATURAL INITIATION OF INFECTION  ? ERADICATION ?  VIRULENT ORGANISMS ISOLABLE FROM _ LIVER, SPLEEN OR OTHER ORGANS  MULTIPLE NODU(COMPLETE BREAKDOWN. .LAR ABSCESSES, OF HOST CONSOLIDATION DEFENSES)  V  (NORMAL RESPONSE IN RESISTANT HOST)  (CORTISONE, LEUKEMIA OR OTHER)  V  LATENT OR CONTROLLED INFECTION (IF FEWER ORGANISMS THAN STATED)  J  DEATH OF HOST  t  ABSCESSES IN KIDNEY CORTEX OR CASEOUS ABSCESSES IN MESENTERIC NODES  t  INJECTION OF OVERWHELMING NUMBERS SENSITIVE MICE 10^ RESISTANT MICE 10?  F i g . 20. 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B i o l . and Med. 85_: 5 1 7 - 5 2 1 .  F u r t h e r problems of lymphocyte N.Y. Acad. S c i . l l j : 8 6 7 - 8 8 6 .  113 APPENDIX I C a l c u l a t i o n of RLV ID^Q u s i n g the Reed and Muench Method f o r d e t e r m i n i n g 5 0 $ endpoint ( 6 6 ) Accumulative  * Log o f Virus Dilution  Mice i n f e c t e d / Mice Intotal used fected  Percentage of Mice Mice Mice total not i n - i n not i n -Toinfected fected fected t a l fected  -1  23/27  23  4  46  4  50  92  -2  13/26  13  13  23  17  40  57  -3  7/27  7  20  10  37  47  21  -4  2/30  2  28  3  65  68  4  -5  1/7  1  . 6  1  71  72  1  T o t a l o f accumulative percentages:  175  * 1/10 ml i n j e c t e d of each of these d i l u t i o n s . The formula f o r the p r o p o r t i o n a t e d i s t a n c e of the ID^Q below the d i l u t i o n g i v i n g next above 50 p e r cent i n f e c t i o n i s : ( i n f e c t i o n at d i l u t i o n next above) - 50 p e r cent = (infection (infection next above) next below)  Proportional distance  S u b s t i t u t i n g from the t a b l e above i n t o the p r e c e d i n g equation: 57 - 50 = 7 57-21 36  o r approximately 0 . 2  Because the d i l u t i o n s a r e expressed l o g a r i t h m i c a l l y , the p r o p o r t i o n a t e d i s t a n c e can be added d i r e c t l y to the d i l u t i o n next above, as expressed i n the t a b l e : 2.0 .2 2.2 2 2 Thus, when the v i r u s i s d i l u t e d 1 : 1 0 * , 0 . 1 ml c o n t a i n s enough v i r u s to i n f e c t 5 0 $ of the mice i n j e c t e d .  114  APPENDIX I I C a l c u l a t i o n s of DNA Base Ratios- f o r C. k u t s c h e r i s t r a i n s ATCC 1 5 6 7 7 and ATCC 1 5 6 7 8 . ~ D e n s i t y was c a l c u l a t e d u s i n g the f o l l o w i n g formula: *I  p  =  /  g  +4.2 6 J  2  (r - r 2  ) x IO  2  g cm"  - 1 0  where / % = d e n s i t y of standard DNA  (E. c o l l ,  Q  r At 4 , 6 0 0  = d i s t a n c e of standard DNA from c e n t e r of r o t a t i o n = d i s t a n c e o f sample DNA from center of r o a t i o n  r a d i a n s / s e c , and u s i n g 1 . 7 1  E. c o l l DNA standard, to II  1.71)  ( 4 4 , 0 0 0 rev/min = 4,600 radians/sec)  CO = speed i n r a d i a n s / s e c r  3  as the d e n s i t y o f the  the above formula  can be s i m p l i f i e d  the f o l l o w i n g : p  0  = 1.71  + 0.0089  ( r - r ) g cm" 2  2  D i s t a n c e o f absorpt i o n peaks from the center of r o t a t i o n :  Sample 1:. E.  3  coll  6.13  C. k u t s c h e r l ATCC 1 5 6 7 7  (r)  6.19  Sample 2 : E.  coli  ( o> r  C. k u t s c h e r l ATCC 1 5 6 7 8  (r)  Density o f C. k u t s c h e r i ATCC 1 5 6 7 7 = 1.71 = 1.701  + 0.0089(6.19  2  -  6.13 ) 2  6.84 6.76  115 Density  of C. k u t s c h e r l ATCC 15678 = 1.71  + 0.0089(6.76  2  -  6.84 ) 2  =1.717 The DNA base r a t i o was c a l c u l a t e d u s i n g the f o l l o w i n g formula: = 0.098(GC) + 1 . 6 6 0 g  *III  where  = buoyant d e n s i t y  cm"  3  in g  cm"  (GC) = mole f r a c t i o n guanlse p l u s  3  cytosine  S u b s t i t u t i n g the d e n s i t i e s c a l c u l a t e d above f o r the two sample DNAs f o r  i n e q u a t i o n I I I g i v e s the f o l l o w i n g  results: Buoyant Density  Mole F r a c t i o n Guanine p l u s Cytosine  C. k u t s c h e r l ATCC 1 5 6 7 7  1.701  42.0  C. k u t s c h e r l ATCC 15678  1.717  58.2  * Formulae c i t e d by S c h i l d k r a u t et a l ( 7 3 ) .  116  APPENDIX I I I  •  • .  D e t a i l of D e t e r m i n a t i o n of P h a g o c y t i c I n d i c e s as presented i n Table V I I I 1.  Source o f s e r a  2.  C o n d i t i o n of mouse  3.  Tubes counted  4.  Macrophages counted  5.  B a c t e r i a counted  CF^SPF) normal  normal  WSW RLV  Infected  12  9  10  625  452  512  4,861  6,695  4,220  6 . Average numbers of b a c t e r i a per macrophage  7.8  7.  1.00  P h a g o c y t i c index  WSW  14.8 1.89  8.2 1.05  E x p l a n a t i o n of above terms: 1.  See Table V I I I  2.  See Table V I I I  3 . Tubes counted. T h i s i s the number of separate prepar a t i o n s made under each c o n d i t i o n . 4.  Macrophages counted. Approximately the f i r s t 50 macrophages encountered on m i c r o s c o p i c examination which contained any b a c t e r i a were counted. (Those which had not phagocytosed were not counted because they may not have been v i a b l e ) .  5.  B a c t e r i a counted. T h i s i s the combined t o t a l of a l l b a c t e r i a phagocytosed by a l l the macrophages.counted i n a l l p r e p a r a t i o n s under each c o n d i t i o n .  6 . Average numbers of b a c t e r i a per macrophage. the t o t a l f o r 5 d i v i d e d by the t o t a l 4 . 7.  This i s  P h a g o c y t i c index. The average number of b a c t e r i a i n g e s t e d p e r macrophage i n the column headed CF]_(SPF) was expressed as u n i t y because s e r a used i n t h i s group of tubes was taken from normal, C. k u t s c h e r i f r e e  117  mice. The i n d i c e s i n the remaining columns are expressed as a r a t i o of the above. Thus: WSW  normal  =  WSW  (RLV I n f e c t e d )  =  14.8 __ 1.89 7.8 8.2 7.8  =1.05  118  APPENDIX IV C a l c u l a t i o n of the Average Constant K f o r Clearance of P 3 l a b e l e d C. k u t s c h e r i from the C i r c u l a t i n g Blood o f V a r i o u s Mouse S t r a i n s 2  Mouse WSW  Condition  Cl  Normal  1398  266  1.5  3.5  0.36  2  2219  168  1.2  4.0  0.40  3  1902  426  1.3  3.0  0.38  4  820  197  1.75  3.25  0.41  5  1923  296  1.5  3.5  0.40  6  2417  308  1.2  3.2  0.45  1  c  2  T  T  l  K  2  0.40  Average A/J  1428  272  1.5  3.75  2  1273  234  1.5  3.5  3  1602  360  1.3  3.0  0.38  4  2202  382  1.25  3.25  0.38  5  1820  322  1.5  4.0  0.30  1  Normal  •  Average WSW  1 2 3  RLV Infected  0.32 0.37  0.35  1702  543  1.5  3.0  0.33  837  190  2.0  k.7  0.24  648  71  1.5  4.0  0.38  4  2371  988  1.25  3.25  0.19  5  1869  560  1.25  3.25  0.26  Average  0.28  continued ...  1  1  9  APPENDIX IV - continued T h i s i s s u p p o r t i n g d a t a f o r F i g . 1 ? » where the slope o f the curve, which r e p r e s e n t s the decrease i n the l o g a r i t h m of the numbers of b a c t e r i a i n the c i r c u l a t i n g blood w i t h time, has been drawn a c c o r d i n g to the average K f o r each group of mice, c a l c u l a t e d as above. The f o l l o w i n g equation i s used K = l o g C! - l o g T  2  -  T  i n the above d e t e r m i n a t i o n : C  2  X  where l o g C]_ = l o g of count a t Time T-^ log C T.1  2  = l o g of count a t Time  T  2  time i n min of f i r s t blood sample taken ( a f t e r i n j e c t i o n of p 3 l a b e l e d C. k u t s c h e r i ) . 2  time i n min of second sample taken.  blood  

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