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A study on the blood protozoa of blue grouse on Vancouver Island Woo, Patrick Tung Kee 1964

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A STUDY ON THE BLOOD PROTOZOA OF BLUE GROUSE ON VANCOUVER ISLAND  by  .  PATRICK TUNG KEE WOO B.Sc.  University of B r i t i s h  Columbia, 1962  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE DEGREE OF Master o f Science  i n t h e Department of Zoology  We accept t h i s t h e s i s as conforming  t o the  required standard  The U n i v e r s i t y o f B r i t i s h September, 1964«  Columbia  In p r e s e n t i n g  this thesis i n p a r t i a l fulfilment  of the requirements f o r an advanced defree at the U n i v e r s i t y  of  B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y available f o r reference  and  study. I f u r t h e r agree t h a t  permission  f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes be granted by the Head of my I t i s understood t h a t  Department or by h i s  Department of Zoology,  Vancouver $, September  Canada.  1964.  representatives.  copying or p u b l i c a t i o n of t h i s t h e s i s f o r  f i n a n c i a l gain s h a l l not be allowed without my  U n i v e r s i t y of B r i t i s h  may  Columbia,  written  permission.  ABSTRACT The  present  study  demonstrates t h a t blue grouse on  Vancouver I s l a n d a r e i n f e c t e d with two probably  two  s p e c i e s o f Haemoproteus.  species of Leucocytozoon and  a species  of  Trypanosoma. Haemoproteus dendragapi n.sp. Nanaimo Lakes Area.  The  i s d e s c r i b e d from the  growth r a t e of H.  canachites-gametocytes  i s much more r a p i d than t h a t d e s c r i b e d by F a l l i s The  very young t i s s u e stages  of H. c a n a c h i t e s  i n Ontario.  are d e s c r i b e d from  l u n g p r e p a r a t i o n s of grouse c h i c k s . The  life  cycle of Leucocytozoon bonasae has  completed by u s i n g a new  vectorCnephia  minus.  As  been  reported  by F a l l i s . i n O n t a r i o , Simulium aureum has been found to be ;  v e c t o r of L. bonasae on Vancouver I s l a n d .  T h i s study  v e r i f i e d Woodcock's o f t e n i g n o r e d hypothesis  A probable  new  has  that the morphology  of the gametocyte-host c e l l complex changes with age infection.  a  of  s p e c i e s of Leucocytozoon i s described  from the Campbell R i v e r Area. I n - v i t r o c u l t u r e of the trypanosome from grouse blood has been c a r r i e d out.  A y e a r l i n g blue grouse has been  s u c c e s s f u l l y i n f e c t e d by i n o c u l a t i o n of m e t a c y c l i c from the c u l t u r e .  trypansomes  ACKNOWLEDGEMENTS The  author wishes t o express h i s s i n c e r e g r a t i t u d e t o  Dr. J.R. Adams f o r h i s constant encouragement, e n t h u s i a s t i c guidance, and c r i t i c a l  s u p e r v i s i o n d u r i n g the e n t i r e phase  of t h i s work. Thanks a r e due t o Drs. C.V. Finnegan and J.F. B e n d e l l f o r t h e i r h e l p f u l suggestions during t h e p r e p a r a t i o n o f the manuscript.  The author wishes to thank Dr. C.V. Finnegan f o r  the use of h i s photographic equipment. The Professor  author i s indebted to Dr. P.C.C, Garnham,  o f P r o t o z o o l o g y , London School o f Hygiene,  f o r a c r i t i c a l r e v i e w o f some o f the work. due t o Dr. A.M. F a l l i s ,  Thanks a r e a l s o  Department of P a r a s i t o l o g y ,  Research Foundation, f o r h i s c r i t i c a l  London,  Ontario  comments and a l s o  encouragements. Throughout  a l l phases o f t h e work, the author had  g r e a t l y b e n e f i t e d from t h e a s s i s t a n c e o f o t h e r s . particularly also  This i s  t r u e o f the c o l l e c t i o n o f l i v e i n f e c t e d b i r d s and  ' c l e a n ' grouse c h i c k s .  To Messrs. F.C. Z w i c k e l ,  J.Dixon,  G.G. Gibson and L.J„ R u s s e l l the author i s t r u l y g r a t e f u l f o r t h e i r a s s i s t a n c e i n the f i e l d . To Mr. T. Barsby t h e author wishes t o express h i s g r a t i t u d e f o r t h e use o f h i s cabin i n the Nanaimo Lakes  Area  during t h e summer of 1 9 6 4 . The author a l s o wishes  to thank Mr. A r t h u r Ko, Dept.  o f B a c t e r i o l o g y , f o r h i s a s s i s t a n c e i n making up the c u l t u r e medium f o r the in-=vitro s t u d i e s .  TABLE OF CONTENTS Page INTRODUCTION  MATERIALS AND METHODS Blood Smear P r e p a r a t i o n and S t a i n i n g Study of T i SSV16 St^&^6S  o  e  o  '  I n - v i t r o C u l t u r e Technique Capture o f B i r d s  o  o  o  10  .  o  o  o  e  o  •  o  •  •  XX 12  . . . .  13  . . . . . . . . . . . . . . . . . .  14  Exposure o f B i r d s . . . . . . . . . . . . RESULTS AND DISCUSSIONS X o HgLon o p r o t G i i s  •  0  0  0  0  0  *  0  0  D e s c r i p t i o n of Haemoproteus  0  0  0  0  0  «  o  •  «  «  •  X6  17  dendra&api n.sp  D e s c r i p t i o n of Immature Stages o f H. c a n a c h i t e s  . . .  19  D e s c r i p t i o n o f Tissue Stages o f H. c a n a c h i t e s  . . .  21  .  23  Discussxon  0  0  .  0  I I . Leuco cytozoon  o  .  .  0  0  .  .  0  .  0  .  0  .  0  .  0  .  0  .  .  .  .  L i f e Cycle o f Leucocytozoon bonasae R e l a t i o n s h i p between  0  .  .  .  .  .  .  .  .  .  .  .  .  .  25  .  27  . . . . . . . . .  Game toeyte Age and 33  P a r a s i t e - H o s t C e l l Shape i n L. bonasae D e s c r i p t i o n o f Leucocytozoon 'A  ?  s cu  s s x on  0  0  0  0  0  0  0  0  0  0  0  36  . . . . . . . . . . o  o  o  o  e  o  o  •  •  •  37  TABLE OF CONTENTS  (continued) Page  I I I . Trypan osoma  • ' • . . . . . . . . . . ' . . • • • * •  42  D e s c r i p t i o n o f Grouse Trypanosoma  42  I n - v i t r o C u l t u r e of Trypanosoma and Experimental I n f e c t i o n o f Grouse and Domestic Chicks w i t h C u l t u r e d Forms . . . . . .  43  Discus si on  46  . . . . . . . . . .  SUMMARY AND CONCLUSION LITERATURE CITED  . . . . . . . . .  . . . . . . . . . . . . . . .  . . . . . . . . . . . . .  . . . . . .  .  57 6S  LIST OF FIGURES FIGURE  Page  1.  Key t o the Measurements made on Leucocytozoon  . . .  59  2.  Key to t h e Measurements made on Trypanosoma . . . .  60  3.  Photomicrograph o f Haemoproteus c a n a c h i t e s and H. dendragapi Garnetocytes . . . . . . . . .  61  4.  Photomicrograph o f Immature Male and Female 61  H. dendragapi Gametocytes 5.  Photomicrograph o f Mature H. dendragapi Gametocytes  6.  Photomicrograph o f of Leucocytozoon Photomicrograph o f of Leucocytozoon  7.  Attenuated bonasae . Attenuated bonasae .  Form . . . Form . . .  (Type . . . (Type . . .  62  I) . . . . . II) . . . . .  63 63  Photomicrograph o f Round Leucocytozoon bonasae 9.  Photomicrograph o f Female Leucocytozoon 'A'  10»  Photomicrograph o f Tissue Stages of  GcLlHGtOCyt©  e  o  o  o  o  Haemoproteus c a n a c h i t e s  o  o  o  o  o  o  o  o  o  a  o  o  o  *  . . . . . . . . . . . . .  11.  Photograph o f Hooded B i r d besides C o l l e c t i n g Cage .  12.  Photograph o f F i e l d Equipment f o r the C o l l e c t i o n and Maintenance of Engorged F l i e s . . I l l u s t r a t i o n o f E x f l a g e l l a t i o n o f Male L , bonasae Gametocyte i n Stomach o f B l a c k F l y . . . . . . .  13.  65 66 66 67  14.  I l l u s t r a t i o n o f Zygote o f L . bonasae i n Stomach of Black F l y . . . . . . . .  67  15.  I l l u s t r a t i o n o f Ookinete o f L.bonasae i n Stomach of B l a c k F l y . . . . . . . . . . . . . .  67  16.  I l l u s t r a t i o n o f S p o r o z o i t e of L. bonasae i n S a l i v a r y Gland o f Black F l y . . . . . . . . . .  67  LIST OF TABLES TABLE  Page  I.  Summary o f P a r a s i t i c Protozoa Observed'in A d u l t and Y e a r l i n g BDue Grouse i n Vancouver I s l a n d  II.  Blood Smears o f Blue Grouse Chicks C o l l e c t e d at the 1963 Game Check . . . .  22  III.  P a r a s i t e Stages Recovered from D i s s e c t e d F l i e s a f t e r Engorgement ."•  29  IV.  Summary of Experimental T r a n s m i s s i o n o f L.Bonasae to Blue Grouse and Domestic Chicks . 7 . . . * .  30  Record of Engorged F l i e s C o l l e c t e d from Exposure B i r d  31  V. VI. VII.  Comparison o f V a r i o u s S i z e s of Leucocytozoon GctniG t o cy 16 s o o a o o © o o o « « o © • <> «  «  / , . 10a  «  S e q u e n t i a l Smears Showing the R e l a t i o n s h i p between Garnetocyte Age and Form i n Leuco cyt ozoon bonasae . . . . . . . . . . . . . .  V I I I . Summary of Experimental Work on Blue Grouse Trypanosomes . . . . . . . . . . IX.  X.  Comparison of Blue Grouse Trypanosomes with Measurements o f Trypanosoma avium and T. g a l l i n a r u m as p u b l i s h e d i n L i t e r a t u r e . . . . . . Summary o f Measurements and Ratios of Blue Grouse Trypanosomes and those of the C u l t u r a l Fo  •  3^  35a 47  43  INTRODUCTION T h i s r e s e a r c h p r o j e c t was undertaken as an attempt to i d e n t i f y the p a r a s i t i c blood p r o t o z o a o f the blue grouse, Dendragapus obscur us f u l i g i n o s u s  T  (Ridgway), i n Vancouver  Island. Fowle (1946) r e p o r t e d t h e occurrence of Haemoproteus •.sp., Leucocytozoon sp., and Trypanosoma sp« from two l o c a l i t i e s i n Vancouver  Island .  He gave f i g u r e s on the i n c i d e n c e and  the degree o f i n f e c t i o n i n 44 b i r d s examined i n 1943-44. Adams and B e n d e l l  (1953) i n d i c a t e d t h a t o f 252 a d u l t b i r d s  examined from Campbell R i v e r , Vancouver o c c u r r e d i n 92%  ?  I s l a n d , Haemoproteus  Leucocytozoon i n $7%, and Trypanosoma i n 76%,  B e n d e l l (1955), r e p o r t e d s i m i l a r h i $ i  protozoan i n f e c t i o n s i n  y e a r l i n g blue grouse.' H i s study was c a r r i e d out i n the s p r i n g and summer o f 1950, 1951, and 1952. examined, 169 had Haemoproteus [97%), [&5%),  and 134 had Trypanosoma (70$).  Of the 174 y e a r l i n g s 14# had Leucocvtozoon C h i c k s examined  d u r i n g t h i s same p e r i o d showed a lower percentage of i n f e c t i o n (Haemoproteus 66%- Leucocytozoon 33%, and Trypanosoma 20%), f  None o f these p r e v i o u s workers had attempted to i d e n t i f y these b l o o d p r o t o z o a t o s p e c i e s .  The main purposes  of t h i s t h e s i s ares (1)  to e s t a b l i s h  the i d e n t i t i e s o f the b l o o d p a r a s i t e s  i n the Vancouver  I s l a n d b l u e grouse, and t o d e s c r i b e  new species" i f they o c c u r .  -2= (2)  to i d e n t i f y the v e c t o r s r e s p o n s i b l e f o r the t r a n s m i s s i o n o f these p a r a s t i i c protozoa, and also the time o f t r a n s m i s s i o n . P a r a s i t i c blood protozoa  v a r i o u s grouse. are  have been r e c o r d e d  from  The f o u r most f r e q u e n t l y r e p o r t e d genera  (a) Haemoproteus, (b) Leucocytozoon. (c) Plasmodium  f  and  ( d) Trypanosoma.. (a) Haemoproteus The  e a r l i e s t r e c o r d of Haemoproteus i n grouse was  that o f Sambon ( 1 9 0 $ ) .  He named i t Haemoproteus mansoni. but  no d e s c r i p t i o n was g i v e n .  Fatham (1910) working w i t h r e d  grouse (Lagopus s c o t i c u s )  again r e c o r d e d  H. mansoni.: he found  what he thought were young stages and a very b r i e f d e s c r i p t i o n was g i v e n .  Clarke  (1 93 3 ), and F a l l i s  (1945) r e p o r t e d a h a l t e r  -shaped Haemoproteus from r u f f e d grouse i n O n t a r i o . and Bennett  Fallis  ( i 9 6 0 ) d e s c r i b e d and named a h a l t e r - s h a p e d  s p e c i e s from spruce  grouse  (Canachites  canadensis) H. c a n a c h i t e s .  In B r i t i s h Columbia. Haemoproteus i n f e c t i o n s i n blue grouse were r e p o r t e d by Fowle Bendell  (1946),  Adams and B e n d e l l  (1953), and  ( 1 9 5 5 ) ; but none o f these r e p o r t s i n d i c a t e what  s p e c i e s was  present.  F a l l i s and Bennett  ( i 9 6 0 ) s t u d i e d t h e stages o f  sporogony o f H. c a n a c h i t e s i n C u l i c o i d e s sphagnumensis W i l l i a m s , and were able to i n f e c t the l i f e  cycle.  ' c l e a n ' r u f f e d grouse c h i c k s to complete  They found t h a t t h e mature gametocytes  e x f l a g e l l a t e d w i t h i n three minutes a f t e r they were i n g e s t e d by C u l i c o i d e s . and t h a t the zygotes  were present i n t h e  -3-  stomach twelve hours a f t e r i n g e s t i o n . diameter.  Oocysts  These average 5 . 5 y t i i n  (averaging 6.3/u) were present i n t h e  stomach w a l l f o u r days a f t e r  i n g e s t i o n , and one day l a t e r  d e v e l o p i n g s p o r o z o i t e s were seen i n a few o o c y s t s .  By the  s i x t h day a f t e r i n g e s t i o n o f the blood meal many o o c y s t s T h i r t y o o c y s t s averaged 9.5/u  contained sporozoites.  and  c o n t a i n e d r e s i d u a l bodies 3 - 4 / i i n diameter. The  development  o f t h e t i s s u e stages i n the grouse  was not f o l l o w e d or d e s c r i b e d by these a u t h o r s . In the  the genus Haemoproteus the gametocytes  r e d blood cel.lsand a r e u s u a l l y h a l t e r shaped.  occur i n Schizogony  takes p l a c e i n the lungs, and not i n t h e e r y t h r o c y t e s . known v e c t o r s a r e l o u s e - f l i e s and Wood 1 9 5 7 ;  Fallis  (Huff 1932) and midges  and Bennett 1 9 6 0 K  g e n e r i c name i s H a l t e r i d i u m Labbe, 1&94*  The  (Fallis  A synonym o f t h i s Members o f t h i s  genus a r e extremely common i n w i l d b i r d s and a l s o i n domestic pigeons, ducks and t u r k e y s .  Coatney  (1936) gave a check  and h o s t - i n d e x o f t h e s p e c i e s o f Haemoproteus  f  list  and Herman  (1944), l i s t e d the s p e c i e s r e p o r t e d from North American  birds.  (b)\Leucocytozoon Leucocytozoon l o v a t i Seligmann and Sambon 1903 was first  d e s c r i b e d from t h e r e d grouse  Sambon (190$). the  Fantham  mature gametocyte  immature gametocyte  (Lagopus  (1910) gave a f u l l e r  d e s c r i p t i o n of  stages and he also d e s c r i b e d t h e  stages.  C l a r k e (1935) d e s c r i b e d  Leucocytozoon bonasae from r u f f e d grouse and spruce grouse  s c o t i c u s ) by  (Bonasa  umbellus)  (Canachites canadensis) i n Algonquin Park,  -4- . Ontario.  He had e a r l i e r noted the occurrence of t h i s p a r a s i t e i n  Algonquin Park, and i t s a s s o c i a t i o n with a heavy m o r t a l i t y of young b i r d s  (Clarke 1934). F o l l o w i n g i t s d e s c r i p t i o n , L.bonasae  was r e c o r d e d by F a l l i s grouse.  (1945) and E r i c k s o n (1953) from r u f f e d  Cowan and P e t e r l e  sharp t a i l e d  (1957) r e p o r t e d i t i n Michigan  grouse.  No attempt t o s o l v e the l i f e when F a l l i s and Bennett r u f f e d grouse  c y c l e was  made u n t i l  (195$) s u c c e s s f u l l y i n f e c t e d  195$  'clean'  c h i c k s by i n j e c t i n g a s a l i n e s u s p e n s i o n of  s p o r o z o i t e s from b l a c k f l i e s t h a t had p r e v i o u s l y f e d on i n f e c t e d grouse. The v e c t o r s r e s p o n s i b l e f o r the t r a n s m i s s i o n of L.bonasae i n Algonquin Park are Simulium l a t i p e s and S. aureum. . . F a l l i s and Bennett  (1962) f o l l o w e d the sporogony  o f L. bonasae i n b l a c k  f l i e s and compared t h e v a r i o u s stages with those from L.mirandae Franca 1912  and L. f r i n g i l l i n a r u m Woodcock 1910.  that the sporogony  They found  qf some i n d i v i d u a l s was completed  than f i v e days a t 22°C+2;  in less  o t h e r s r e q u i r e d nine days or more.  Oocysts of L. mirandae averaged 14/u  (9-19) i n diameter,  o f L. bonasae 13yu ($-16), and those of L. f r i n g i l l i n a r u m ($-16).  those llyu  No measurements were g i v e n f o r the s i z e of t h e  s p o r o z o i t e s except t h a t i t was  noted t h a t the s p o r o z o i t e s of  L. mirandae were s l i g h t l y longer and narrower the other two  than those of  s p e c i e s and more numerous i n the l a r g e r o o c y s t s .  F a l l i s and Bennett  (195$) had noted that the  gametocytes  of L. bonasae c i r c u l a t i n g i n the b l o o d of the b i r d were of two forms:  one i n which the gametocyte i s i n a host  cell  ' c l e a n ' grouse: a grouse t h a t has been r a i s e d as a c h i c k i n * an indoor cage and the b l o o d found t o be n e g a t i v e f a r blood protozoa p r i o r t o i n f e c t i o n .  that has a t t e n u a t e d ends,  the o t h e r form i n a host  c e l l t h a t l a c k s the a t t e n u a t e d ends.  They i n d i c a t e d that  t h i s i s p o s s i b l y due to a d i f f e r e n c e i n gametocyte age. In the genus Leucocytozoon the micrpgametocyte  the macrogametocyte and  o c c u r i n the l e u c o c y t e s or, i n some  s p e c i e s , a l s o i n the e r y t h r o c y t e s . Schizogony  takes p l a c e i n  the parenchyma o f t h e l i v e r , h e a r t , kidney or other organs, the s c h i z o n t s forming l a r g e bodies d i v i d e d into  cytomeres.  There i s no schizogony i n the e r y t h r o c y t e s or l e u c o c y t e s . The vectors are black f l i e s Bennett 1 9 5 6 ; Skidmcre Leucocytozoon  (O'Roke 1 9 3 4 ; F a l l i s , Anderson and 1 9 3 2 ; and F a l l i s and Bennett  i s common i n many w i l d b i r d s and i s the cause  of d i s e a s e i n ducks, geese, t u r k e y s and chckens. (1937)  1953).  Coatney  gave a catalogue and host-index o f the genus, and Herman  (1944) l i s t e d the s p e c i e s o c c u r r i n g i n North American  birds.  (c) Plasmodium Wetmore  (1939) was the f i r s t to r e p o r t a Plasmodium  from the p r a i r i e s h a r p - t a i l e d grouse.  T h i s grouse Plasmodium  i s c h a r a c t e r i s e d by the elongated gametocytes the host  c e l l nucleus  that  displace  s l i g h t l y when mature by producing from  f o u r t o twelve m e r o z o i t e s . was given i n the paper.  No i d e n t i f i c a t i o n of the Plasmodium  Fallis  (1945)  r e p o r t e d a s p e c i e s of  Plasmodium w i t h c h a r a c t e r s resembling those of P. circumflexum i n r u f f e d grouse.  Experimental r e s u l t s p u b l i s h e d a year l a t e r  ( F a l l i s 1946) i d e n t i f y t h i s Plasmodium to be a s t r a i n o f P. circuflexum  ?  and he r e f e r r e d to i t as the grouse Plasmodium.  Dorney and Todd ( I 9 6 0 ) i n a survey of t h e s p r i n g i n c i d e n c e of r u f f e d grouse blood p a r a s i t e s i n W i s c o n s i n a l s o noted  Plasmodium i n f e c t i o n .  The percentage of i n f e c t i o n i s very low  throughout th e year . About f o r t y s p e c i e s of Plasmodium have been d e s c r i b e d from b i r d s , but only f o u r t e e n o r f i f t e e n are accepted (Hewitt 1940; Bray 1957; L a i r d and L a r i 1953).  catalogues  s p e c i e s o f Plasmodium i n b i r d s .  takes p l a c e i n the  and h o s t - i n d i c e s o f the  The c h i e f c h a r a c t e r i s t i c s o f  t h i s genus are, the gametocytes occur schizogony  Many w i l d  Herman (1944.) and Coatney and  b i r d s are commonly i n f e c t e d . Roudabush (1949) have given  as v a l i d  i n the  erythrocytes,  e r y t h r o c y t e s and a l s o i n v a r i o u s  other t i s s u e s . A l l the three genera mentioned so f a r , belong t o the sub-order Haemosporina.  Chief c h a r a c t e r i s t i c s of t h i s  suborder ares the zygote i s m o t i l e , schizogony the v e r t e b r a t e h o s t , and sporogony i n the the e r y t h r o c y t e s are invaded, from the host c e l l The  pigment  invertebrate. I f  (hemozoin) i s formed  hemoglobin.  gametocytes o f both the genera Haemoproteus and  Plasmodium occur  i n the e r y t h r o c y t e s .  be e i t h e r round or e l o n g a t e d .  These gametocytes can  Plasmodium i s d i s t i n g u i s h e d  from Haemoproteus by the f a c t that  schizogony  the e r y t h r o c y t e s t o produce merozoites; schizogony  takes p l a c e i n  takes p l a c e i n  while i n Haemoproteus  i s r e s t r i c t e d to the parenchyma o f i n t e r n a l organs.  Hence a r t i f i c i a l t r a n s m i s s i o n of Plasmodium i s p o s s i b l e by blood i n n o c u l a t i o n w h i l e Haemoproteus can be e x p e r i m e n t a l l y t r a n s m i t t e d only by t i s s u e t r a n s p l a n t s o r by i n n o c u l a t i o n o f t i s s u e emulsion.  (d) Trypanosoma Trypanosomes have been r e p o r t e d under a l a r g e number of names from many s p e c i e s of b i r d s . a l i k e and  probably  cross transmission relationships.  They a l l look very much  belong to r e l a t i v e l y few  species.  Extensive  s t u d i e s are needed to e s t a b l i s h t h e i r  They are however r e f e r r e d to i n t h i s t h e s i s  by the names under which they were f i r s t Trypanosoma avium Danilewsky,  described. was  first  d e s c r i b e d from owls ( s p e c i f i c name not given) and ( C o r a c i a s g a r r u l u s ) i n Europe, and has  roller-birds  s i n c e been r e p o r t e d  from a wide v a r i e t y of b i r d s i n c l u d i n g crows (Baker, 1956) Canada geese (Diamond and Herman 1954)...  Baker  (1956  a,  t r a n s m i t t e d i t from the rook (Corvus f r u g l e g u s ) and the (J3.  monedula) t o c a n a r i e s , but f a i l e d t o transmit  s i n g l e 3-day-old  chicken  chick.  jackdaw  __ described  i n South-East A s i a ; i t i s about 3^-u l o n g .  g a l l i n a r u m Bruce e t a l . , 1911 in  b)  i t to a  T. c a l m e t t e i Mathis and.Leger, 1909 was from the  and  was  C e n t r a l A f r i c a as about 60  d e s c r i b e d from the  long.  T. hannai was  chicken described  from the pigeon, and T. numidae from the guinea f o w l . Avian a t t a i n i n g great longer. the is  The  trypanosomes are very polymorphic, sometimes size.  be 26yuto 6C^u l o n g or even  k i n e t o p l a s t i s g e n e r a l l y a l o n g d i s t a n c e from  p o s t e r i o r end. often  They may  There i s a f r e e f l a g e l l u m , and  the body  striated. Blood-sucking  T.  arthropods such as mosquitoes  and  -3h i p p o b o s c i d s are b e l i e v e d t o be v e c t o r s o f a v i a n trypanosomes, but  t h e o n l y complete l i f e  a,  c y c l e was worked out by Baker (1956,  b) f o r T. avium from rooks and jackdaws.  England the h i p p o b o s c i d f l y , the  vector.  He found t h a t i n  Ornithomyia a v i c u l a r i a  T  a c t s as  Upon i n g e s t i o n with the b l o o d meal, the  trypanosomes change i n t o the: c r i t h i d i a l form i n the mid-gut, m u l t i p l y by b i n a r y f i s s i o n i n t h i s form, and pass t o the h i n d gut.  They m u l t i p l y f u r t h e r  and then t u r n i n t o a p i r i f o r m  stage which develops i n t o a s m a l l m e t a c y c l i c trypanosome form. B i r d s become i n f e c t e d when they eat i n f e c t e d i n s e c t s .  The  m e t a c y c l i c trypanosomes penetrate the membrances o f the mouth, oesophagus and/or crop and probably invade the lymphatic system, d e v e l o p i n g i n t o l a r g e forms which f i r s t blood 13 to 24 hours a f t e r  appear i n the  infection.  A c c o r d i n g to Baker  (1956), there i s no m u l t i p l i c a t i o n  i n t h e a v i a n h o s t , the trypanosomes simply become l a r g e r . c o u l d account f o r t h e i r  sparse numbers i n the b l o o d .  This  They  p e r s i s t i n the rook and jackdaw over w i n t e r , being more or l e s s r e s t r i c t e d to the bone marrow, and reappear i n the p e r i p h e r i a l b l o o d i n the s p r i n g . too,  Diamond and Herman (1954),  found t h a t T. avium could be i s o l a t e d from the bone  marrow of Canada geese much more r e a d i l y than from the b l o o d . Nothing i s known o f the p a t h o g e n i c i t y o f the avian trypanosomes.  They are presumably non-pathogenic.  Bennett of  (1961) r e p o r t e d T. avium from a g r e a t v a r i e t y  b i r d s i n Algonquin Park i n O n t a r i o .  H i s c o n c l u s i o n s were  t h a t T. avium has a p o s t e r i o r s t a t i o n i n the f l y v e c t o r , and  -9experiments i n d i c a t e d t h a t the p a r a s i t e i s probably  transmitted  by the p e n e t r a t i o n of f l a g e l l a t e s  ( i n the f e c e s of i n f e c t e d  f l i e s ) through breaks i n the host  skin.  =10-  MATERIALS AND /METHODS Blood Smear P r e p a r a t i on an d S t a i n i n g Blood smears f o r m i c r o s c o p i c s t u d i e s o f the  gametocyte  stages were made from b l o o d taken from the b r a c h i a l v e i n o f l i v e b i r d s ; blood from dead b i r d s was taken from the l u n g s . The  smears were a i r d r i e d as r a p i d l y as p o s s i b l e to prevent  any change i n the form o f the p a r a s i t e b e f o r e the smears have d r i e d , s i n c e i t i s known t h a t mature microgametocytes  of  t•  Leucocytozoon may temperature  e x f l a g e l l a t e w i t h i n a minute a t room  ( F a l l i s and Bennett 1953)  and Haemoproteus gametocytes temperature i s lowered For  may  and immature Leucocytozoon  round up when the b l o o d  (Woodcock 1910).  quick d i a g n o s i s o f p a r a s i t i c i n f e c t i o n i n the  f i e l d , d r i e d b l o o d f i l m s were s t a i n e d with Wrightfe S t a i n f o r two minutes, a f t e r which an equal volume o f b u f f e r e d water  (pH. 7.0)  minutes.  was  added and l e t stand f o r another  T h i s was f l u s h e d o f f w i t h water  2-3  and the smear d r i e d  and examined under medium power f o r trypanosomes Leucocytozoon.  distilled  For Haemoproteus the smear was  and  examined under  oil. For  d e t a i l e d study o f p a r a s i t e morphology the  smears vere f i r s t f i x e d i n 100$ methyl a l c o h o l f o r one t o two minutes, d r i e d and then s t a i n e d w i t h Giemsa S t a i n f o r approximately f o r t y - f i v e minutes. (Shute and Maryon 1961) for  was  better d i f f e r e n t i a t i o n .  The  s a l i n e Giemsa technique  s l i g h t l y m o d i f i e d by t h i s author Sodium c h l o r i d e  (0.5 gm)  d i s s o l v e d i n one l i t r e o f b u f f e r e d d i s t i l l e d water  \  (pH  was 7.2).  -11The s t a i n i n g medium i s made up o f 0.8 c c . o f "Gurrs Giemsa S t a i n  Improved  ( R 6 6 ) " i n 45 c c . o f Q.005% NaCl b u f f e r e d  distilled  water . The smears were s t a i n e d f o r a t l e a s t f o r t y minutes a f t e r which no change o c c u r r e d .  Smears l e f t i n the s t a i n f o r  twenty-four hours appeared as i f they have been s t a i n e d f o r f o r t y minutes. Study o f T i s s u e Stages (Schizogony i n Host T i s s u e s ) Three d i f f e r e n t approaches were used i n s t u d y i n g the  t i s s u e stages o f Leucocytozoon and Haemoproteus i n the  host b i r d .  1. liver,  They were: 1.  Organ Smear.  2.  Organ  Impression.  3.  Organ  Section.  Organ Smear.  A minute piece of host t i s s u e (e.g.  s p l e e n or lung) was mashed on a clean s l i d e , a t the  same time s p r e a d i n g i t as t h i n l y as p o s s i b l e .  I t was then  f i x e d i n 100% methyl a l c o h o l and s t a i n e d i n Giemsa's s t a i n as d e s c r i b e d 2.  earlier.  Organ Impression. T h i s was made by i m p r i n t i n g the  f r e s h l y cut s u r f a c e s of an organ on to a c l e a n s l i d e .  This  was f i x e d i n 100% methyl a l c o h o l and s t a i n e d as p r e v i o u s l y described. 3.  Organ S e c t i o n .  and Cooper  The method used was t h a t o f S h o r t t  (194?) w i t h s l i g h t m o d i f i c a t i o n s .  F r e s h organs  taken from b i r d s were c u t into p i e c e s two t o t h r e e c e n t i m e t e r s t h i c k and f i x e d immediately i n Carnoys F i x a t i v e f o r approxi m a t e l y twenty-four h o u r s .  -12-  CarnoysFixative: 30 c c .  G l a c i a l Acetic Acid  30 c c .  .Chloroform  30 c c .  Absolute A l c o h o l  organ s l i c e s were then t r a n s f e r r e d to 95% a l c o h o l and  The  f i n a l l y to 70% a l c o h o l where t h e y were preserved u n t i l sectioning.  The organ s l i c e s were s e c t i o n e d  microns t h i c k .  at f o u r to f i v e  In the s t a i n i n g t e c h n i q u e , d i f f i c u l t y i n  d i s s o l v i n g the colophonium i n acetone t o the recommended concentration in  as l i t t l e  soluble  was  overcome by f i r s t  d i s s o l v i n g the  a b s o l u t e a l c o h o l as p o s s i b l e  colophonium  (colophonium i s very  i n a b s o l u t e a l c o h o l ) , and adding t h i s t o the acetone.  A l s o 2 c c . o f "Gurr's Giemsa" was  used i n s t e a d of the 10 c c .  as recommended. I n - v i t r o C u l t u r e o f Trypanosoma In-vitro  c u l t u r e o f trypanosomes  from the blue grouse  was c a r r i e d out i n the d i p h a s i c medium used by Tobie, von Brand, and Mehlman (1950) f o r the c u l t i v a t i o n of T. gambiense and T. rhodesiense. base  T h i s medium i s composed o f a r i c h blood agar  ( s o l i d phase) and a l i q u i d phase i n which the  trypanosomes  divide. The c u l t u r e medium was  first  i n c u b a t e d at 37C f o r  twenty-four hours t o ensure that, the medium was f r e e of b a c t e r i a l contamination. 64) was  then i n o c u l a t e d  The whole procedure was  B l o o d from the i n f e c t e d grouse  (S4-  i n t o the l i q u i d phase o f the medium. c a r r i e d out as a s e p t i c a l l y as p o s s i b l e .  To f u r t h e r ensure t h a t the c u l t u r e s were f r e e of b a c t e r i a l  -13 =  contaminations 1500 u n i t s o f p e n i c i l l i n was added to each o f the c u l t u r e t u b e s . The  c u l t u r e s were incubated  a t 25°C f o r s i x days and  checked every twenty-four hours t o f o l l o w t h e course o f the d i v i d i n g forms i n the c u l t u r e s . l i v e preparations  These forms were s t u d i e d i n  as w e l l as i n f i x e d and s t a i n e d smears.  The  smears were heat d r i e d and then s t a i n e d with Giemsa's s t a i n . A f t e r s i x days o f i n c u b a t i o n c u l t u r e s were incubated at  25°C.  at 25°C, f o u r o f the s i x  a t 3$°C, while two c u l t u r e s were  The c u l t u r e s were l e f t  at t h e i r respective  left  new  temperatures f o r another f o u r days checking every twenty-four hours f o r s i g n s o f b a c t e r i a l c o n t a m i n a t i o n s . On the tenth  day o f i n c u b a t i o n  three  o f t h e 3$°C  c u l t u r e s were i n o c u l a t e d i n t r a - p e r i t o n e a l l y i n t o a grouse y e a r l i n g and two young domestic c h i c k s Also old  'clean'  (four weeks o l d ) .  one of the 25°C c u l t u r e s was i n j e c t e d i n t o a f o u r week domestic The  chick. e x p e r i m e n t a l b i r d s were checked f o r the presence  o f S-shaped trypanosomes by d i r e c t blood stained preparations.  examinations and by  /  T h i s was done twenty-four and f o r t y -  e i g h t hours a f t e r i n f e c t i o n .  Also  blood  from these  inoculated  b i r d s was c u l t u r e d at 25°C to c o n f i r m i n f e c t i o n . F i e l d Methods:-  Capture of B i r d s  L i v e i n f e c t e d blue grouse were caught w i t h a l o n g pole  (12-15 f e e t ) that has a l o o s e noose at one end.  u s u a l l y done with the h e l p were o b t a i n e d  o f a t r a i n e d dog.  Grouse  This  was  chicks  from the w i l d broods a few days a f t e r they had  -14hatched when t h e i r capture was f e a s i b l e , u s u a l l y d u r i n g the l a s t two weeks o f June and the f i r s t two weeks of J u l y . F o l l o w i n g capture, b l o o d smears were made from each o f them to  determine whether they were i n f e c t e d p r i o r t o c a p t u r e .  Blood smears were made a t r e g u l a r  i n t e r v a l s from i n f e c t e d  c h i c k s t o f o l l o w the course of i n f e c t i o n . Exposure Method (Anderson 1956) The i n f e c t e d b i r d was p l a c e d i n a cage  (14"xl4"xl4").  A hood, made of dark c l o t h p l a c e d over t h e head o f the exposed b i r d keeps the b i r d r e l a t i v e l y q u i e t i n the exposure cage so t h a t f l i e s are able  t o f e e d on i t w i t h o u t being d i s l o d g e d . The  b i r d i n t h e exposure cage can be r a i s e d t o v a r i o u s h e i g h t s by means of a rope. The cage with the hooded b i r d was r a i s e d t o the d e s i r e d h e i g h t i n ten t o f i f t e e n seconds and l e f t there f o r approximately t h i r t y minutes.  I t was then lowered c a r e f u l l y  (30-40 seconds) on t o a plywood square (22'x2^ ) f o l l o w i n g f  which i t was covered w i t h a c o l l e c t i n g cage which c o n s i s t e d of a frame the  covered with screens on f o u r  sides;  t o p b e i n g covered w i t h a p l a s t i c sheet and the bottom  open. the  (20"x20"x20")  left  S t r i p s o f rubber sponge glued t o the bottom frame o f  cage f o r m a t i g h t s e a l and prevent the escape o f f l i e s .  The b i r d was p l a c e d i n the c o l l e c t i n g cage f o r approximately t h i r t y minutes.  Engorged f l i e s which had l e f t  the b i r d and  s e t t l e d on t h e s i d e o f the cage were c o l l e c t e d i n a s p i r a t o r s through a s l e e v e a t the s i d e o f t h e c o l l e c t i n g cage.  -15= B i r d s were exposed a t tiie Nanairao Lakes Area d u r i n g the  period  June 12th t o J u l y 1 s t .  Unpublished r e c o r d s from  p r e v i o u s years (Gibson) show t h i s p e r i o d t o be the h e i g h t o f the  b l a c k f l y season i n t h i s a r e a .  The b i r d s were u s u a l l y  exposed i n the evenings between 6.30 and 9.30 p.m. a t a h e i g h t of  f o r t y f e e t , but on very windy evenings t h e exposure h e i g h t  was f i f t e e n  feet.  The  engorged  f l i e s were kept i n s m a l l cages  (Anderson 1956) i n which a h i g h h u m i d i t y and f r e e water were p r o v i d e d by wet paper towels p l a c e d a t the bottom o f the cage. L o n g e v i t y o f the f l i e s was presumably r e d u c i n g the l i g h t  and the r a t e o f e v a p o r a t i o n i n the cages  by means o f l o o s e l y f i t t i n g d i l u t e honey.  extended by  covers.  F l i e s were f e d d a i l y on  Specimens were d i s s e c t e d i n 0.&5% s a l i n e a t  r e g u l a r i n t e r v a l s a f t e r t h e i r engorgement (Shute arid Maryon I960), heat f i x e d and then p l a c e d i n a b s o l u t e methyl  alcohol.  This d i s s o l v e d the sodium c h l o r i d e c r y s t a l s before the p r e p a r a t i o n s were s t a i n e d i n Giemsa. Heads and thoraces of f l i e s ,  i n which  sporozoites  were presumed t o be present were e m u l s i f i e d i n Qo$5% s a l i n e and i n j e c t e d subcutaneously i n t o a ' c l e a n ' y e a r l i n g b l u e grouse. Fly  emulsions were a l s o i n j e c t e d with s a l i n e i n t o one week o l d  domestic c h i c k s . saline.  As c o n t r o l s , some b i r d s were i n j e c t e d with  Blood smears were prepared from a l l experimental  b i r d s to determine i f they had become i n f e c t e d , and i f so, t o f o l l o w the i n f e c t i o n i n th e b l o o d .  -16=  RESULTS S t u d i e s o f blue grouse c h i c k s , y e a r l i n g s , and a d u l t s from t h r e e l o c a l i t i e s on Vancouver I s l a n d d u r i n g the summers of 1963 and 1964 have confirmed (1946),  Adams and B e n d e l l  (1953)  p r e v i o u s r e c o r d s o f Fowle (1955),  and B e n d e l l  :|  Vancouver I s l a n d blue grouse are i n f e c t e d w i t h  blood  that protozoa  of three genera, namely Haemoproteus, Leucocytozoon,, and Trypanosoma. D e t a i l e d s t u d i e s o f the b l o o d smears o f b i r d s from each of these l o c a l i t i e s i n d i c a t e that there are two s p e c i e s of Haemoproteus one  T  probably  two s p e c i e s of Leucocytozoon and  species o f Trypanosoma.  Haemoproteus Of the two s p e c i e s of Haemoproteus i d e n t i f i e d as H. c a n a c h i t e s F a l l i s  one has been  T  and Bennett I960.  i d e n t i f i c a t i o n i s based p u r e l y on t h e m o r p h o l o g i c a l  This similar-  i t i e s o f the gametocyte stages as found i n the blood o f our Vancouver; I s l a n d blue grouse and the s p e c i e s H. c a n a c h i t i e s d e s c r i b e d by F a l l i s i n O n t a r i o .  T h i s s p e c i e s i s common to a l l  b i r d s i n the three study a r e a s .  The percentage o f i n f e c t i o n  of y e a r l i n g and a d u l t b i r d s d u r i n g the s p r i n g and summer o f both 1963 and 1964 was about 93$ (Table I ) .  Blood  smears of  chicles made from t h e 1963 Game Check a t Campbell R i v e r i n d i c a t e only 2 6 . 9 $ i n f e c t e d a t the end of August  (Table I I ) . .However  a c h i c k caught i n l a t e August near Courtenay s t a r t e d t o show the i n f e c t i o n on August 2 9 t h , and a d e t a i l e d d e s c r i p t i o n o f the v a r i o u s stages o f gametocyte f o r m a t i o n was  obtained.  =16 a TABLE  I  Summary o f P a r a s i t i c Blood Protozoa observed i n A d u l t and Y e a r l i n g Blue Grouse (1963-64)  Date  Locality  June 1963  Middle Quinsam Lake  July 1963  I n f e c t e d B i r d s Sample S i z e Haemoproteus Leucocytozoon Trypanosoma  Middle Quinsam Lake Courtenay  ./  11  11  11  !0  9  9  9  9  12  12  12  10  August 1963  Middle Quinsam Lake  6  6  6  March 1964  Courtenay  1  0  1  May 1964  Courtenay  3  2  3  June 1964  Nanaimo Lake Area  20  20  20  16  62  60  62  54  100%  S3.9%  TOTAL; • %  Infected  96.. 3%  1  ' 3 '""  -17The other Haemoproteus r e c o r d e d from t h e Vancouver I s l a n d blue grouse d u r i n g the study p e r i o d i s a new s p e c i e s . The name Haemoproteus dendragapi i s proposed f o r i t . range  of t h i s new s p e c i e s seems r a t h e r l i m i t e d .  The  I t occurs i n  grouse i n the Nanaimo Lakes Area, but b i r d s from the other two study areas are i n f e c t e d only w i t h H. c a n a c h i t e s .  Pure  i n f e c t i o n o f H. dendragapi i s found i n grouse up B l a c k Jack Ridge Nanaimo Lakes Area  (3 out of 4)0  Blue grouse that are  found i n t h e f l a t s below B l a c k Jack Ridge have a mixed i n f e c t i o n o f H. c a n a c h i t e s and H. dendragapi Haemoproteus dendragapi n. s p . Host:  ( F i g u r e s 4 and 5)  Dendragapus obscurus f u l i g i n o s u s .  Locality:  Black Jack Mountain,  (18 out o f 18),.  (Ridgway)  Nanaimo Lakes Area, Vancouver  Island. S l i d e s used i n the f o l l o w i n g d e s c r i p t i o n a r e A9, A10, D22. EARLY GAMETOCYTES Form and S i z e :  S p h e r i c a l or s l i g h t l y ovate, appearing as s i n g l e h y a l i n e o b j e c t s between the nucleus of the r e d blood c e l l and i t s p e r i p h e r y . S i z e about 1-1.5/u„  Double i n f e c t i o n i n a s i n g l e host c e l l Nucleus:  i s not uncommon.  Not d i f f e r e n t i a t e d , r e p r e s e n t e d by a p e r i p h e r a l blob o f s t a i n i n g r e d chromatin.  Cytoplasm:  C l e a r , p e r i p h e r y darker with o c c a s i o n a l  reticular  areas which s t a i n pale b l u e . Vacuole:  A s m a l l c l e a r area occupying almost the e n t i r e organism  Pigment Granule:  Absent .  -18-  . .IMMATURE-GAMETOCYTES Form and S i z e :  (Figure 4) .  S p h e r i c a l or s l i g h t l y l o c a t e d at one  ovate b o d i e s , u s u a l l y  end o f the l o n g a x i s o f the r e d  blood c e l l nucleus.  Average s i z e 1.5-4.5/u  diameter; s l i g h t l y  d i s p l a c i n g the nucleus o f  the r e d b l o o d c e l l  to one  side i n older stages.  Male gametocyte s t a i n i n g blue while  female  stains intensely blue. Nucleus:  More d e f i n i t e l y r e c o g n i z a b l e as a d i s t i n c t  structure.  U s u a l l y mare c e n t r a l l y l o c a t e d i n o l d e r s t a g e s . Oval to  s p h e r i c a l i n shape and s t a i n i n g r e d e s p e c i a l l y  around  the p e r i p h e r y . N u c l e a r membrane not s h a r p l y  defined. Cytoplasm:  V a r y i n g from c l e a r  to g r a n u l a r , blue s t a i n i n g  r e a c t i o n much more pronounced than i n e a r l i e r s t a g e s . Vacuole:  Vacuole now d i f f u s e or broken up i n t o 2-3 s m a l l e r v a c o u l e s ; d i f f u s e d or absent i n o l d e r s t a g e s .  Pigment Granule:  From 3-16, o c c u r r i n g i n groups o f 2-4. May be i r r e g u l a r l y s c a t t e r e d , approaching those of the a d u l t gametocyte i n s i z e .  MATURE GAMETOCYTE  ( F i g u r e 5),  Male Gametocyte Form and S i z e :  S l i g h t l y ovate to s p h e r i c a l ; d i s l o d g i n g the red blood c e l l n u c l e u s completely t o one s i d e and f r e q u e n t l y occupying the former space o f the r e d b l o o d c e l l cytoplasm c o m p l e t e l y . Length 6.84-11.2/a Width 5-75-3.04/1  Nucleus:  Cytoplasm: Vacuole:  Ovate to s p h e r i c a l , can be anywhere i n the p a r a s i t e cytoplasm. S t a i n s pink i n Giemsa. Average s i z e 1 . 5 3.5yU becoming more d i f f u s e d . Karyosome not u s u a l l y seen • P a l e , almost  hyaline.  Very d i f f u s e d and sometimes i n d i s t i n c t .  -19-  Pigment  Minimum number 1 9 , maximum number 3 0 ;  Granule:  tendency to be i n groups o f 3 - 5 ; shape from s p h e r i c a l t o r o d - l i k e ; carbon black i n appearance, h i g h l y r e f r a c t i l e . Host C e l l :  E n l a r g e d ; and only t h e host c e l l n u c l e u s can be seen, the space f o r m e r l y occupied by host c e l l protoplasm i s a l l taken up by the p a r a s i t e gametocyte.  Female Gametocyte Form and S i z e :  L i k e male gametocyte;  ovate to  spherical  d i s l o d g i n g r e d b l o o d c e l l n u c l e u s to one s i d e , hence g r e a t l y d i s f i g u r i n g the r e d blood c e l l . Measurements i n g e n e r a l are the same as f o r male gametocyte, but t h e r e i s g r e a t e r tendency to produce greater hypertrophy o f the r e d b l o o d cell. Nucleus:  Spherical  to o v a l , more compact than that Average s i z e 1-2.5/A  gametocyte.  of male  diameter, s t a i n s  dark pink to r e d ; karyosome f r e q u e n t l y d i s t i n c t  and  i s u s u a l l y on the p e r i p h e r y o f the n u c l e u s . Cytoplasm:  •Vacuole : Pigment  E a s i l y d i s t i n g u i s h a b l e from that of male gametocyte by t h e darker s t a i n i n g . R e t i c u l a r appearance q u i t e distinct. Not commonly seen.  Granule:  Host C e l l :  As i n male  gametocyte.  As i n microgametocyte.  D e t a i l e d D e s c r i p t i o n o f the Immature Stages of H. c a n a c h i t e s Blue grouse c h i c k , "NOBAND I I " , was approximately 45  days o l d when i t was  Quinsam Area, Vancouver on August 2 3 r d , made on August  c a p t u r e d on August 2 2 n d Island.  and put i n t o 23rd,  t  i n the Middle  I t was brought back to  an indoor cage.  Vancouver  Blood smears  2 5 t h , and 2 7 t h , showed i t to be f r e e of  -20-  blood p r o t o z o a .  On August 2 9 t h a t 1 0 . 0 0 A.M.  smear was  T h i s smear has very young r i n g stages between  made.  the nucleus  o f the r e d b l o o d c e l l  and  a f o u r t h blood  i t s periphery.  They  look not u n l i k e the r i n g stages of Plasmodium and i n f a c t were mistaken  to be such at that time.  r i n g s are measured.  Most of them are s l i g h t l y o v a l ,  average l e n g t h being 2.2/0 diameter  of the  Twenty-one of  cytoplasm  reticular absent.  p a r a s i t e nucleus  i s about 0.56yu .  i s c l e a r , the  This  staining red.  Pigment g r a n u l e s  are  vacuole i s a s m a l l c l e a r a r e a .  The This  the  p e r i p h e r y i s darker with o c c a s i o n a l  areas which s t a i n pale b l u e . The  such  and the width b e i n g 1.46yli ; the  n u c l e u s i s j u s t a p e r i p h e r a l b l o b of chromatin The  they  fifth  made a t 2 . 0 0 P.M.  smear was  smear, made four hours a f t e r  29th.  on August  the p r e v i o u s smear shows an }  i n c r e a s e i n the t r o p h o z o i t e s i z e ; these older t r o p h o z o i t e s are q u i t e e l o n g a t e d .  Eighteen  average length i s i+.02j(x and more d i s t i n c t and of  specimens are measured and  the width  2.8/tt .  The nucleus i s  measures about 1.2^ i n diameter.  these t r o p h o z o i t e s (7 out of IB)  the  A number  has y e l l o w pigment i n the  p a r a s i t e cytoplasm,  and i n some t h e r e are 2 or 3 dark pigment  granules a l o n g with  the y e l l o w ones.  The  s i x t h smear was  29th.  In t h i s  size.  Male and female  on August  smear the gametocytes have grown to about  full  gametocytes can be d i s t i n g u i s h e d by  their staining reactions. blue and  made a t 1 0 . 0 0 P.M.  The female  gametocytes s t a i n dark  i n some the karyosome can' be i d e n t i f i e d «  gametocytes s t a i n l i g h t blue and the nucleus  The  i s rather  male  -21-  elongated and d i f f u s e d . to  Though these gametocytes have grown  a d u l t s i z e they can be d i s t i n g u i s h e d from the s e x u a l l y  matured ones, by being h i g h l y vacuolated, by s t i l l y e l l o w pigments along with very f i n e The gametozoites  having  dark pigment g r a n u l e s .  seventh smear made on September 2nd shows these t o be f u l l y matured.  V a c u o l a t i o n has disappeared,  the y e l l o w pigments have given way t o the u s u a l dark  refractile  pigment granules. Tissue Stages  of Haemoproteus c a n a c h i t e s ( F i g u r e 10)  Lung smears ( K l ; and K26) made from two blue  grouse  c h i c k s ( f i e l d number 6 3 - 6 ; and 63-16 r e s p e c t i v e l y ) show very young t i s s u e stages o f H. c a n a c h i t e s . at  These smears were made  the 1963 Game Check held a t Campbell R i v e r (Table I I ) .  Smear K l shows a very l i g h t Haemoproteus i n f e c t i o n , while K26 i s negative f o r blood s t a g e s .  Because o f the immaturity o f  the t i s s u e stages,the host c e l l s to  t h e macrophage system.  can be i d e n t i f i e d as belonging  Manwell (1961) and Kudo ( i 9 6 0 ) have  s t a t e d t h a t the t i s s u e stages o f Haemoproteus occur mainly i n the e n d o t h e l i a l system of the l u n g s . The youngest  stage from these  smears i s a minute  u n i n u c l e a t e body w i t h i n the cytoplasm o f t h e host c e l l .  This  minute u n i n u c l e a t e body i s composed o f a s i n g l e r e d chromatin dot w i t h dark blue s t a i n i n g cytoplasm.  T h i s probably  r e p r e s e n t s the f i r s t  stage a f t e r the entry o f the s p o r o z o i t e  i n t o the host c e l l .  As t h e p a r a s i t e grows, the r e d s t a i n i n g  nucleus s t a r t s d i v i d i n g u n t i l t h e r e are about twelve of these d i s t i n c t n u c l e a r masses.  At t h i s phase of growth, d i v i s i o n  -22TABLE  II  Game Check Blood Smears - B3ue Grouse Chicks, 1963. Field NO.  Slide NO.  T L  o  ™ n ^ H y , «. c  a  l  i  t  T.  H  63^- 6  Kl  Beaver Lk, /  / /  63 -9  K2  Gooseneck Lk./  - -  63 -11 K3 63 -15 K4  Bacon Lk.  -  63.-18 K5 L.Quinsam L k . 63 -30 K6 63 - 3 2 K7 63 -34 K8  M e r r i l l Lk. Camp V Mohum Lk.  /  63 - 3 8 K9 Mohum Lk. 63 -49 K 1 0 Campbell Lk.63 -56 K12 Campbell L k . / 63 -63 K13 Camp V. 63 -67 K14 Gooseneck L k r 63 -73 K15 P a t t e r s o n Lk.63 -74 K16 Gooseneck Lk./ 63 -82 K17 M.Quinsam Lk.63-102 K18 B e r r y Creek/ 63--107K19 63 -108 K20  CampV. Camp V.  /  K22 63 - 2 7 K23 L.Quinsam Lk. 63 -12 K24 Bacon Lk. K25 K26  T i s s u e s t a t e s o f Haemoproteus: a t t e n u a t e d L. bonasae. Heavv i n f e c t i o n o f H. c a n a c h i t e s . Heavv i n f e c t i o n of L. bonasae (attenuated)  / /  - -  Menzies Mt. -  Negative. Heavv i n f e c t i o n o f L.bonasae (attenuated)  / / / / / / / / / / / / / / / / / / / / / / /  Remarks  Heavv i n f e c t i o n of L.bonasae (attenuated) Attenuated L. bonasae Young of H.canachites: a t t e n u a t e d Leucocytozoon. Attenuated L . bonasae.  /  -  35  Attenuated L. bonasae. Attenuated L. bonasae.  /  -  56  Leucocytozoon"A" and L. bonasae. Leucocytozoon"A" and L . bonasae.  / / /  L i g h t i n f e c t i o n o f L. bonasae. L i g h t Haemoproteus and L. bonasae.  -  Leucocytozoon "A" and L . bonasae. L i g h t Haemoproteus and L. bonasae.  / / / / / / / /  Attenuated L. bonasae. Leucocytozoon "A" and L . bonasae. Attenuated L. bonasae Attenuated L. bonasae Round L. bonasae.  K  3 5  L i g h t L . bonasae ( a t t e n u a t e d ) . Leucocytozoon"A" and L. bonasae t i s s u e s t a g e s of Haemoproteus.  Percentage i n f e c t i o n of c h i c k s a t the end of August, 1 9 6 3 : Leucocytozoon  2  ^/26  Haemoproteus Trypanosoma  x  2  /26  0  0  " 9 »3$ 2  x 100 - 26.9%  7/ 6 l g  1  x  1  0  0  =  T  also  -23-  of the nuclear masses s t o p s , while growth i n s i z e c o n t i n u e s . Each of these nuclear masses i s now c a l l e d a cytomere.  The  o l d e s t s t a g e found so f a r i s t h e twelve cytomere stage i n which f i v e of t h e twelve cytomeres show s i g n  of budding.  These f i v e cytomeres r e p r e s e n t the b e g i n n i n g of the next and f i n a l stage i n the schizogony o f the p a r a s i t e . Discussion Haemoproteus  dendragapi ( F i g u r e s 4 and 5) i n s p i t e  of i t s round gametocytes i s c o n s i d e r e d a Haemoproteus  because  no s c h i z o n t s were ever d e t e c t e d i n blood smears of 21 b i r d s that harboured the i n f e c t i o n .  These b i r d s were k i l l e d a t  v a r i o u s times of day over a p e r i o d of 25 days i n June.  Also  the s t a i n i n g r e a c t i o n o f H. dendragapi i s i d e n t i c a l to that of H. c a n a c h i t e s (Figure 3) (which has the u s u a l h a l t e r gametocytes).  T h i r d l y , the presence of r e f r a c t i l e  pigment  granules that are s i m i l a r to those of H. c a n a c h i t e s make i t more of a Haemoproteus. 1905, a Haemoproteus  F o u r t h l y H. s a c h a r o v i Now  and MacNeal,  with rounded gametocytes has been  confirmed by Huff (1932) who  worked out the l i f e  cycle.  The most prominent f e a t u r e of H. dendragapi i s the manner i n which the mature gametocyte pushes the nucleus of the r e d b l o o d to one s i d e so t h a t the l a t t e r appears t o s i t on the p a r a s i t e  as a cap.  T h i s s p e c i e s d i f f e r s g r e a t l y from  H. canachites i n form and s i z e . from immature  A l l the gametocyte stages,  to a d u l t forms, are round while the  forms of H. c a n a c h i t e s are e l o n g a t e d and the adult h a l t e r shape.  The number o f pigment granules  immature gametocytes  i n the a d u l t  -24-  gametocytes of both s p e c i e s i s approximately H. dendragapi  the same.  resembles H. s a c h a r o v i i n that  both  have round gametocytes which push the r e d blood c e l l n u c l e i to  one s i d e .  They d i f f e r  greatly i n size  (H. dendragapi i s  smaller than H. s a c h a r o v i ) i n the number o f pigment (H. dendragapi and f i n a l l y  has many more pigment granules than  i n host b i r d  (H. dendragapi  granules H.sacharovi).  i s d e s c r i b e d from  blue grouse and H. s a c h a r o v i from mourning  doves).  R e s u l t s from Npband I I show t h a t the t r o p h o z o i t e s of  H. canachites from our Vancouver I s l a n d blue grouse grow  to  the adult gametocyte stage w i t h i n 12 hours.  T h i s growth  r a t e i s f a r g r e a t e r than t h a t r e p o r t e d by F a l l i s and Bennett (I960) f o r H. c a n a c h i t e s i n t h e i r e x p e r i m e n t a l l y i n f e c t e d r u f f e d grouse.  According t o F a l l i s  takes approximately mature s i z e .  and Bennett  (I960) i t  6 days f o r the t r o p h o z o i t e t o grow t o  This g r e a t d i f f e r e n c e i n t h e rate o f growth of  the gametocyte i s hard to account might c o n t r i b u t e towards t h i s  for.  V a r i o u s f a c t o r s which  great d i f f e r e n c e i n growth r a t e  have been c o n s i d e r e d but none seemed s a t i s f a c t o r y . made on August 2 7 t h was  The smear  (two days b e f o r e r i n g stages were seen)  c a r e f u l l y examined under an o i l immersion lens f o r stages  younger than those r e c o r d e d from the smear made on the 2 9 t h , at 1 0 . 0 0 A.M., but no p a r a s i t e could be f o u n d . of  In f a c t none  the smears made b e f o r e the 2 9 t h show any s i g n of an  infection.  -25-  The  a d u l t gartEtocytes o f H. c a n a c h i t e s from the  r u f f e d grouse  i n O n t a r i o are v e r y s i m i l a r  i f not i d e n t i c a l to  the h a l t e r shape H. c a n a c h i t e s found i n our blue Because of the great s i m i l a r i t i e s  grouse.  of the gametocytes our  h a l t e r shape Haemoproteus has been t e n t a t i v e l y i d e n t i f i e d • as H. c a n a c h i t e s .  Further i n v e s t i g a t i o n by c r o s s i n f e c t i o n s  and  the study of t i s s u e stages s h o u l d c o n f i r m i t s i d e n t i t y . The young t i s s u e s t a g e s from the lung smears of 2 c h i c k s were i d e n t i f i e d as those o f H. c a n a c h i t e s : they resemble the i l l u s t r a t i o n s and d e s c r i p t i o n s of the t i s s u e stages of H. columbae i n Kudo's "Protozoology".  The  stages cannot  p o s s i b l y be t h o s e o f Leucocytozoon  because i n  Leucocytozoon  there is always a c e n t r a l body present i n a  developing megaloschizont of Leucocytozoon liver  (Cowan 1 9 5 5 ) .  Also,  tissue  megaloschizonts  develop mainly i n the s p l e e n , h e a r t , and  (Cowan 1 9 5 5 ) ;  and -foe t i s s u e stages of Haemoproteus  develop mainly i n the e n d o t h e l i a l c e l l s I 9 6 0 , Manwell 1 9 6 1 ) .  of the lungs  (Kudo  This author has r e p e a t e d l y found  fully  grown Haemoproteus s c h i z o n t s from the lungs of juncos. As c o n f i r m a t i o n , t i s s u e stages s i m i l a r to those d e s c r i b e d from the c h i c k s have a l s o been recorded from lung smear of a y e a r l i n g shot i n e a r l y Nanaimo Lakes A r e a .  the  June 1964 at the  A blood smear from t h i s p a r t i c u l a r  a l s o shows very young r i n g stages i n the r e d b l o o d  yearling  cells.  Leucocytozoon V a r i o u s forms of Leucocytozoon  were encountered  blood smears from b i r d s during the study p e r i o d .  in  F i g u r e s 6,  7,  -26g, and  9 are photomicrographs o f the gametocytes of  Leucocytozoon t h a t are found i n the c i r c u l a t i n g b l o o d .  Figure  1 shows the b a s i s on which they were measured. I n i t i a l l y i t was  not known whether these forms  were v a r i a n t s of a s i n g l e s p e c i e s or m  only through a study o f sequence  d i f f e r e n t s p e c i e s , and  of b l o o d smears from i n f e c t e d  b i r d s , by comparisons o f smears from b i r d s i n d i f f e r e n t l o c a l i t i e s and by l i f e c y c l e s t u d i e s were the f i n a l  conclus-  i o n s reached. As s t a t e d e a r l i e r , two s p e c i e s of Leucocytozoon are present i n the Vanoo uver I s l a n d blue grouse. species i d e n t i f i e d i n this  Of the  two  study, one o f them corresponds  c l o s e l y to the d e s c r i p t i o n o f Leucocytozoon bonasae Clarke 1935.  Hence i t i s r e f e r r e d t o as such i n t h i s t h e s i s .  two y e a r l i n g and adult  b l u e grouse, examined from the t h r e e  study areas between 1963 t h i s form (100$ from the 1963  and 1964,  were a l l p o s i t i v e s f o r  i n f e c t i o n , Table I ) .  Game Check (Table  percentage of i n f e c t i o n  Sixty  (92.3$).  B l o o d smears of c h i c k s  I I ) a l s o show a very high A l l o f f i v e c h i c k s shot on  J u l y 2 3 r d near Courtenay were p o s i t i v e f o r L. bonasae.  These  chicks were a l l from d i f f e r e n t broods i n the same g e n e r a l area.  Another one of these chicks was  caught  and brought  back to the l a b o r a t o r y where the course of i n f e c t i o n o f the blood s t a g e s was  studied.  The other Leucocytozoon s p e c i e s r e c o r d e d i n t h i s study resembles the d e s c r i p t i o n o f L. f r i n g i l l i n a r u m Woodcock 1910,  i n form o n l y .  I t i s d i s t i n c t l y 2-3  longer.  S i n c e only  -27s e x u a l l y matured forms were found, i t i s d i f f i c u l t positive i d e n t i f i c a t i o n .  to make a  It i s in a l l likelihood a  new  s p e c i e s , but u n t i l more i s known o f i t s v a r i o u s blood stages and the v e c t o r s r e s p o n s i b l e f o r i t s t r a n s m i s s i o n , i t w i l l t e n t a t i v e l y be r e f e r r e d t o as Leucocytozoon Leucocytozoon  'A*.  *A' seems q u i t e l o c a l i z e d .  only been found i n the Campbell R i v e r A r e a .  I t has  Blood smears o f  c h i c k s from the Game Check show that about 20% were i n f e c t e d with t h i s Leucocytozoon  (5 out o f 26; Table I I ) . No  i n f e c t i o n of Leucocytozoon  pure  'A'has been r e c o r d e d so f a r , i t  u s u a l l y comes a s a mixed i n f e c t i o n with Leucocytozoon  bonasae.  I t has not been r e c o r d e d from y e a r l i n g s and a d u l t s i n the Campbell River Area d u r i n g the summers of 1963  and  1964.  L i f e Cycle of Leucocytozoon bonasae An adult female blue grouse 28th, 1964 i n Courtenay was  (S4-64) caught on  used as the exposure b i r d .  smears made b e f o r e and a f t e r the exposure p e r i o d  May Blood  (June 13th -  J u l y 1st) showed i t to be i n f e c t e d w i t h L. bonasae, H.canachites. and trypanosomes.  Table V i s a f i e l d r e c o r d of the number  and specie s of engorged f l i e s c o l l e c t e d from the exposure b i r d d u r i n g the exposure p e r i o d s a t the Nanaimo Lake Area i n Vancouver  Island. The most abundant  ornithophlic black f l i e s i n t h i s  area d u r i n g the exposure p e r i o d were Simulium aureum and minus.  For a l l p r a c t i c a l purposes a l l b l a c k f l i e s  Cnehpia  collected  from the exposure b i r d are c o n s i d e r e d as b e l o n g i n g t o one o f these two  species.  -28-  Engorged  Simulium,aureum and  Cnephia minus d i s s e c t e d  12 hours a f t e r c o l l e c t i o n had both zygotes and m o t i l e ookinetes (Table I I I ) . The  zygotes average  12.4/1 i n diameter; the  ookinetes a r e c r e s c e n t shaped w i t h one p o i n t e d than the o t h e r . cytoplasm pale b l u e . l  The  The  and more  nucleus s t a i n s p a l e r e d and the  The n u c l e u s , which  l o c a t e d more towards the p o i n t e d end. i n the cytoplasm.  end narrower  is s l i g h t l y oval, i s  There are 2-3  vacuoles  average l e n g t h o f the ookinetes i s 26 /a.  S a l i v a r y glands d i s s e c t e d out of the head of both S. aureum and C,  minus 10 days a f t e r c o l l e c t i o n from the  exposure grouse had s p o r o z o i t e s . 10.4/u i n l e n g t h and l,6Sjix  The  s p o r o z o i t e s are about  i n width.  The n u c l e u s which i s  s l i g h t l y e l o n g a t e d (1.68x1,12/0 s t a i n s r e d while the cytoplasm s t a i n s pale b l u e .  One  end of the s p o r o z o i t e i s more p o i n t e d  than the o t h e r , and the nucleus i s l o c a t e d more to the  rounded  end. On June 28th the heads and t h o r a c e s of two C. minus were e m u l s i f i e d i n 0.85$ s a l i n e and  injected  into  (Table I V ) .  a ' c l e a n ' y e a r l i n g blue grouse  were c o l l e c t e d from the exposure  intra-peritoneally The  2 C.minus  grouse 10 days p r e v i o u s l y  (on June 1 9 t h ) . Blood smears made from t h i s y e a r l i n g grouse 3h weeks l a t e r were p o s i t i v e f o r L. bonasae cycle.  Attempts  (Table I V ) .  t  to i n f e c t domestic  thus completing the  life  chicks w i t h L. bonasae were made.  Ten 2 week o l d domestic c h i c k s were i n o c u l a t e d  with i n f e c t e d f l y suspensions at -various times d u r i n g J u l y .  TABLE  III  P a r a s i t e Stages r e c o v e r e d from D i s s e c t e d F l i e s a f t e r  F l y Species Dissected  Gnephia minus  Cnephia minus  Time a f t e r Engorgement  1 hour  12 hours  Simulium aureum Simulium aureum n u. . Cnephia minus  Simulium aureum  Cnephia minus  P a r a s i t e Stage Found L.bonasae Exflagellation F i g u r e 13 L.bonasae  zygotes ookinetes F i g u r e s 14 & 15  • , 5 - 6 days  leptomonad s t a g e s o f trypanosome  10 days  L. bonasae sporozoites F i g u r e 16  10 days  L. bonasae sporozoites F i g u r e 16  Engorgement  S i t e o f P a r a s i t e Stage  Stomach o f f l y  Stomach of f l y  Fore gut and v i c i n i t y of s a l i v a r y g l a n d  Salivary  gland  Salivary  gland  x  TABLE  IV  Summary o f E x p e r i m e n t a l T r a n s m i s s i o n o f L. bonasae to Blue Grouse and Domestic Chicks  No. o f Birds  Experimental Bird  Age o f Bird  1  Blue grouse  Yearling  3  Domestic chick  2  2  2  No. of F l i e s Time s i n c e Date S p e c i e s of F l i e s fed Injected F l i e s i n j e c t e d i n j e c t e d to each b i r d on i n j e c t e d grouse C. minus  2  10 days  June 28  Remarks  Gametocytes in circulating blood a f t e r 0,5 davs  15 days  C. minus  2  9 days  July 6  Domestic chick  17 days  S. aureum  3  10 days  July 8  Domestic chick  21 days  S. aureum  4  8 days  July  Domestic chick  24 days  C. minus  2  9 days  J u l y 14th N e g a t i v e  12  Negative  . Negative  Negative  o  -31TABLE  V  Record of Engorged F l i e s c o l l e c t e d from Exposure B i r d (June 13-July 1, Date o f Exposure  Time of Exposure  1964)  Weather Condition  Number and Species of F l i e s (engorged) c o l l e c t e d  Very windy 55°F  4 Culicoides  2 C. minus  8 Culicoides  4 C. minus 8 &. aureum  June  13th  1900- -2030  14th 15th  Rain  1830- -2030  Windy 57°F  16th  Rain  17th 18th  Rain  1830- -2030  Calm, very humid,68°F  19th  1830- -2030  20th  1830- -2030  Calm,humid 65°F Calm 70°F  21st  1900- -2030  Very windy 56 °F  26th  1800- -2100  Calm 65°F  27th  1830- -2130  Windy 67°F  28 th  1900- -2130  29 th 30 th July  1st  6 19 21 C u l i c o i d e s 8 9 17 C u l i c o i d e s 10 16 15 C u l i c o i d e s  C. minus S.aureum C. minus S.aureum C. minus S. aureum  3 Culicoides  2 C. minus 4 S. aureum  14 C u l i c o i d e s  9 C. minus 15 S • aur eum  5 Culicoides  5 C. minus 6 S.aureum  Calm 69°F  16 C u l i c o i d e s  9 C. minus 8 S. aur eum  1830. -2130  Calm 70°F  18 C u l i c o i d e s 13 C. minus 6 S. aureum  1830- -2130  Calm 71°F  21 C u l i c o i d e s 13 C. minus 3 S.aureum  Calm 70°F  20 C u l i c o i d e s 15 C. minus 3 S.aureum  1830. -2130  TABLE VI Comparison  Parasite  Leucocytozoon bonasae  Form  Attenuated Form Type I  o f V a r i o u s S i z e s of Leucocytozoon  Gametocytes  Sample Size  Parasite Length (yu)  Parasite Width (^n)  Host Nucleus Host Nucleus Length (/ii) Width (/u)  P a r a s i t e-Host c e l l complex l e n g t h ( yu)  26  21.84-23.52  5.60-3.92  10.08-14.00  3.92-2.80  31.36-34.72  14.56-19.16  11.76-7.84  10.08-14.00  3.92-1.68  25.20-28.56  30  9.52-11.76  11.76-8.96  8.96-13.44  3.92-1.68  35  11.20-12.88  11.76-8.40  6.16-8.40  6.I6-4.48  Figure 6 Attenuated Form Type I I . 25 Figure 7 Round Form  . Leucocy tozoon A'  Figure 8 Round Form  !  Figure 9  -33Some were i n o c u l a t e d w i t h C. minus suspensions and some w i t h S.aureum suspensions.  Blood smears from these c h i c k s were  negative f o r p a r a s i t e s .  This would tend to i n d i c a t e t h a t  L. bonasae i s q u i t e host s p e c i f i c , s i n c e both the grouse and the domestic  chicken a r e g a l l i f o r m  birds.  R e l a t i o n s h i p between Gametocyte Age and P a r a s i t e - H o s t C e l l Shape i n Leucocytozoon. (1958) had noted that the  F a l l i s and Bennett  gametocytes o f L . bonasae c i r c u l a t i n g i n the b l o o d of the b i r d were o f two forms. host c e l l  One, i n which the gametocyte i s i n a  t h a t has a t t e n u a t e d ends, t h e o t h e r i n a host  t h a t l a c k s the attenuated ends.  cell  They i n d i c a t e d t h a t t h i s might  p o s s i b l y be due to a d i f f e r e n c e i n gametocyte age. In  the present study these d i f f e r e n t gametocyte  forms were again r e c o r d e d .  The two forms were  especially  prominent i n a d u l t s and y e a r l i n g s i n May and e a r l y June. Chicks shot i n l a t e June (5 out o f 5) a t the Courtenay  study  area  had only the a t t e n u a t e d forms, while c h i c k s examined l a t e r on i n the summer had both forms.  In s t u d y i n g through the s e r i e s  of b l o o d smears f r o m about t h i r t y c h i c k s c o l l e c t e d between J u l y and August  (most of the smears a r e from the 1963 Game  Check, Table I I ) i t was n o t i c e d t h a t t h e r e was c o n s i d e r a b l e v a r i a t i o n i n l e n g t h and width measurements of both the p a r a s i t e i t s e l f and the p a r a s i t e - h o s t c e l l form.  For convenience  complex i n the a t t e n u a t e d  i n d e s c r i p t i o n s the a t t e n u a t e d forms  are a r b i t r a r y s u b - d i v i d e d i n t o two t y p e s .  -34Type I : - Where t h e p a r a s i t e - h o s t  c e l l complex measures  over 30yu; and t h e p a r a s i t e i t s e l f i s o v a l . (Figure 6 and Table V I ) . Type I I : - Where the p a r a s i t e - h o s t than 30yu  c e l l complex i s l e s s  i n l e n g t h ; and the p a r a s i t e i t s e l f i s l e s s o v a l ,  approaching t h e shape o f t h e round form. (Figure 7 and Table V I ) . Gametocytes that are d e s c r i b e d  as belonging  to the  round form (Figure #) i n t h i s t h e s i s a r e q u i t e round; and are found i n host c e l l s lacking. and  i n which the a t t e n u a t i o n  i s visually  The host c e l l n u c l e u s s t a i n s r e d i n Giemsa's s t a i n  i s l a t e r a l l y compressed t o one s i d e o f the p a r a s i t e - h o s t  complex.  In most o f the specimens s t u d i e d , the o r i g i n a l host  c e l l membranes can be seen as l o o s e l y f i t t i n g  envelopes around  the p a r a s i t e s . "Noband I"was c a p t u r e d near Courtenay on J u l y 2 3 r d , 1963.  The approximate age a t t h e time o f capture was about  30 days.  A blood  smear made a t the time o f capture i n d i c a t e d  that i t was i n f e c t e d w i t h L. bonasae and trypanosomes.  Blood  smears were made on subsequent days to f o l l o w the course o f i n f e c t i o n o f L. bonasae. On s L i d e H63, made on J u l y 2 3 r d , the whole smear contained  only twelve L. bonasae gametocytes, a l l o f which are  of the attenuated  forms.  i t s e l f i s very o v a l  In t e n of these,  (average l e n g t h 22.54/u, width 4 . 6 4 / 1 ) ,  and the whole p a r a s i t e - h o s t 32.#4/u.  the p a r a s i t e  In the other  c e l l complex i s on the average  two specimens the p a r a s i t e i t s e l f i s  -35-  less  oval  (average l e n g t h 17.46/Uwidth 7.94/0 while the  parasite-host c e l l the  gametocytes  complex averages 26.74/U i n l e n g t h .  Hence  j u s t d e s c r i b e d can be c l a s s i f i e d under the  two c a t e g o r i e s of the a t t e n u a t e d forms proposed e a r l i e r as ten  of Type I and two  of Type I I .  In s l i d e J2 made on J u l y 30th (made 7 days H63)  most of the a t t e n u a t e d forms  are of Type I I .  after  Altogether  there are twenty two a t t e n u a t e d forms, of these only f i v e o f the  specimens are of Type I , the remainder seventeen belong  to Type I I . On August 2nd a t h i r d smear was made ( s l i d e J 7 ) . In t h i s smear o n l y Type I I o f t h e a t t e n u a t e d forms are seen. A l s o appearing i n the smear are round forms i n which the h o s t c e l l a t t e n u a t i on i s v i s u a l l y specimens  lacking.  Of the n i n e t e e n  counted, f o u r are of the round form.  p a r a s i t e length i n the round form  The  average  i s 10.12^u and the width i s  9.24/u The l a s t smear was the  chick died.  made on August  5th,  just before  In t h i s smear ( s l i d e J16) t h e r e a r e  predomin-  a n t l y round forms; twenty one of t h e s e specimens a r e round forms w h i l e only f i v e are Type I I of the a t t e n u a t e d form. Autopsy of the c h i c k showed i t to be h e a v i l y w i t h Pispharynx (13) and the p r o v e n t r i c u i u s was and s w o l l e n .  I t was  stopped e a t i n g .  infected  b a d l y damaged  f o r c e d f e d s i n c e August 1 s t when i t  TABLE  VII  S e q u e n t i a l Smears showing the R e l a t i o n s h i p between Game t o c y t e A g e a n d F o r m i n L e j ^ o ^ t ^ z o o n bonasae.  Date  Slide Number  Total No.of Gametocytes  July  23  H63  12  July  30  J2  22  A t t e n u a t e d Forms T y p e 1 T y p e IT  10  Round Forms  2  0  17  0  August  2  J7  19  0  15  August  4  J16  26  0  5  21  -36D e s c r i p t i o n of Leucocytozoon  'A'  (Figure  As mentioned e a r l i e r i n the i n a l l p r o b a b i l i t y a new  the  introduction, this i s  s p e c i e s , but as only s e x u a l l y mature  forms were found i t i s f e l t for  d e s c r i p t i o n of a new Leucocytozoon A T  f  that t h i s  i s i n s u f f i c i e n t data  species. has  been recorded  grouse c h i c k s i n the Campbell R i v e r Area. 1963  Game Check at Campbell River  chicks  (23%)  9).  only from  Records from  blue the  show t h a t s i x o f twenty-six  i n a random sample from t h i s area are i n f e c t e d  with t h i s s p e c i e s .  Blood smears of c h i c k s , y e a r l i n g s ,  a d u l t s from the other two study areas are negative  and  for this  p a r t i c u l a r Leucocytozoon. The  f o l l o w i n g i s a d e s c r i p t i o n of the  on the measurements of t h i r t y - f i v e specimens.  species  based  In Table VI  the measurements of t h i s s p e c i e s are compared w i t h those of L. bonasae C l a r k e , which occurs i n a l l of the three areas i n Vancouver  Island.  Leucocytozoon Woodcock 1910 host  'A'  the host  More important  i s the f a c t that  than macrogametocytes.  and  vacuolated;  the  i n both p a r a s i t e s  s i t s on top o f  Microgametocytes are n o t i c e a b l y  macrogametocyte i s dark blue granular  that  i t d i f f e r s from L . f r i n g i l l i n a r u m  c e l l nucleus i s t r i a n g u l a r and  p a r a s i t e l i k e a 'cap'. frequent  resembles L. f r i n g i l l i n a r u m  i n that both p a r a s i t e s are round and  c e l l lacks attenuation,  in size.  study  The  cytoplasm of  (Giemsa's S t a i n ) , and  the less  the i s quite  t h a t of the microgametocyte s t a i n s  -37-  pale blue and i s more uniform.  The  nucleus of the macrogame-  t o c y t e s t a i n s pale blue and i s more uniform. the macrogametocyte s t a i n s pale r e d and  The  at i t s p e r i p h e r y  i s u s u a l l y a darker s t a i n i n g blob of chromatin, been i d e n t i f i e d as a karyosome (Clarke 1935; The  nucleus  The nucleus  A t c h e l y 1951)* in  cytoplasm  of the  in size.  The  I t i s more d i f f u s e d  p a r a s i t e s themselves are  more or l e s s round and measure 11.20-12.88yu (Table V I ) .  there  of the microgametocyte i s pale r e d ,  o v a l , and i s u s u a l l y c e n t r a l l y l o c a t e d . and i s about 9x3.5/1  of  which has  i s more or l e s s round, about 2.Cyu or 2 , y  s i z e , and can be l o c a t e d anywhere w i t h i n the parasite.  nucleus  by 11.76-8.40yu  As mentioned e a r l i e r the host c e l l l a c k s  attenuations.  I t s nucleus  which l i e s to one s i d e . i s 6.16-8.40/u  i s a dark r e d t r i a n g u l a r body  The  l e n g t h of the host c e l l  , and the height  nucleus  i s 4 . 4 8 - 6 . 1 6 / 1 (Table I I ) .  Discussion In working out the l i f e bonasae F a l l i s  c y c l e of Leucocytozoon  and Bennett (1958) found t h a t the v e c t o r s  r e s p o n s i b l e f o r t r a n s m i s s i o n of the p a r a s i t e i n Park, O n t a r i o , are Simulium aureum and S. Transmission  Algonquin  latipes.  s t u d i e s of L. bonasae. i n the Vancouver  I s l a n d blue grouse, were c a r r i e d out i n the Nanaimo Lakes Area during the p e r i o d June 13th  to J u l y 1 s t .  F i e l d r e c o r d s show  that the most p r e v a l e n t s p e c i e s of o r n i t h o p h l i c black  flies  during t h i s p e r i o d are Simulium aureum and Cnephia minus. D i s s e c t i o n of these engorged f l i e s i n d i c a t e d that sporogony  -38-  of L. bonasae takes p l a c e i n both these as s p o r o z o i t e s were recovered from the  s p e c i e s of black s a l i v a r y glands  d i s s e c t e d f l i e s t h a t had p r e v i o u s l y b i t t e n the C. minus as a new  of  exposure b i r d .  v e c t o r f o r L. bonasae i s f u r t h e r s u b s t a n t i a t e d  by the f a c t t h a t a ' c l e a n ' y e a r l i n g b l u e grouse was with L. bonasae a f t e r i t was suspension  flies  of the heads and  infected  i n o c u l a t e d with a s a l i n e thoraces  o f two  C. minus that  had  b i t t e n the exposure b i r d 10 days p r e v i o u s l y . An a n a l y s i s of the f i e l d r e c o r d shows t h a t of two  s p e c i e s of black f l i e s recovered  the  from the exposure b i r d ,  aureum i s the more abundant i n mid-June and  t h a t C. minus  i s more p r e v a l e n t i n l a t e June and p o s s i b l y e a r l y J u l y . Since both s p e c i e s can act as v e c t o r s , t h i s would mean t h a t S.aureum i s the more important  v e c t o r i n June, and  responsible f o r transmission i n J u l y .  C. minus i s  Hence c h i c k s that  hatch  out d u r i n g June and J u l y would have a good chance of being i n f e c t e d by one  or both the v e c t o r s .  T h i s i s not the  only  i n s t a n c e where t r a n s m i s s i o n of a p a r a s i t e a t d i f f e r e n t times of th e summer i s c a r r i e d out by d i f f e r e n t s p e c i e s of v e c t o r s . Fallis,  Anderson and Bennett (1956) working with  m i s s i o n of L. simondi found t h a t S. c r o x t o n i and euryadiniculum  are the important  p a r t of the b l a c k f l y season (May S. r u g g l e s i i s the important  the  trans-  S.  v e c t o r s during the e a r l y t o June) i n O n t a r i o  v e c t o r i n l a t e June and  R e s u l t s obtained from s t u d i e s o f the  while July.  successive  blood smears (Table VII) made from "Noband I " i n d i c a t e that  -39the  gametocytes i n the attenuated host c e l l s are younger  than those t h a t are i n host c e l l s that seem t o l a c k the attenuation.  T h i s i s shown by the f a c t t h a t blood  smears  made at the beginning of the study p e r i o d had mostly attenuated forms and those t h a t were made a t l a t e r dates had a l l round forms (which i n c i d e n t a l l y do not seem t o show any attenuation).  Since a t t e n u a t e d forms have more o v a l gametocytes  than t h e round forms i t can be s a i d t h a t young gametocytes are o v a l and t h a t o l d gametocytes are round.  This observation  seems t o agree with the view expressed by Woodcock  (1910)  that a u t h o r s " i n d e s c r i b i n g t h e gametocytes (and t h e i r  host  c e l l s ) as rounded, appear t o have been d e a l i n g simply with i n d i v i d u a l s which had begun the a c t i v e process of rounding themselves o f f ,  p r e p a r a t o r y to r u p t u r i n g the host c e l l  and  becoming l i b e r a t e d as r i p e gametocytes". S t u d i e s on the young gametocyte stages of L, bonasae also h e l p to s u b s t a n t i a t e the above c o n c l u s i o n .  Only very  young stages (those t h a t are l e s s than 5/u i n diameter) are round.  As they grow (to about 11.15yu) they become o v a l and  they s t a r t to cause a t t e n u a t i o n o f the host c e l l .  A l s o from  s t u d i e s of very young gametocytes i t i s concluded t h a t mononuclear l e u c o c y t e s are t h e host c e l l s and that are  monocytes  more p a r a s i t i s e d than lymphocytes. The f a c t t h a t only round gametocytes are found i n  a d u l t b i r d s i n l a t e summer, and e a r l y s p r i n g can now  be  e x p l a i n e d as the remnants o f the young gametocytes of the  -40-  previous s p r i n g .  A l s o some o f the c h i c k s from the Game Checks  and some o f t h e c h i c k s shot i n J u l y and August had only the attenuated forms (e.g. 6 3 - 2 2 , 63-49 e t c .  Table I I ) ; t h i s can  be e x p l a i n e d by t h e f a c t that the i n f e c t i o n was a r e c e n t one and t h a t the gametocytes had no time to grow " o l d " . These o b s e r v a t i o n s would tend to r e f u t e the theory t h a t t h e round and a t t e n u a t e d forms are t h e r e s u l t s o f s p o r o z o i t e s and/or merozoites p a r a s i t i z i n g d i f f e r e n t host cells.  A c c o r d i n g t o the reasonings of t h i s  theory one s h o u l d  not expect to f i n d b i r d s w i t h pure i n f e c t i o n s of e i t h e r one form as the p r o p o r t i o n s o f d i f f e r e n t white b l o o d c e l l s i n any one animal should remain r e l a t i v e l y c o n s t a n t .  But i f i t i s  accepted t h a t t h e attenuated forms are t h e younger stages o f the round forms,  then i t i s p o s s i b l e to f i n d b i r d s  with only one form.  infected  A b i r d w i t h a pure i n f e c t i o n of attenuated  forms would i n d i c a t e t h a t the i n f e c t i o n was a r e c e n t one; t h i s i s e s p e c i a l l y t r u e i n c h i c k s c o l l e c t e d i n l a t e summer.  Also  a d u l t b i r d s with a pure i n f e c t i o n of round forms might i n d i c a t e that i t d i d not have a r e c e n t r e l a p s e and t h a t a l l the gametocytes i n t h e blood were "old". A p o s s i b l e e x p l a n a t i o n of t h e r e l a t i o n s h i p between the age of the gametocyte and the shape of the whole p a r a s i t e host c e l l complex i s t h a t as the young o v a l p a r a s i t e grows, more o f the host c e l l cytoplasm  i s used up, and as t h i s  happens the a t t e n u a t i o n on e i t h e r s i d e becomes l e s s pronounced. T h i s goes on u n t i l a l l or most of the h o s t c e l l cytoplasm i s  -41used up.  When t h i s happens a l l or most of the space  o r i g i n a l l y occupied by the host cytoplasm i s now  that  occupied by  the p a r a s i t e , or a l t e r n a t i v e l y t h i s empty space does not show up i n s t a i n e d specimens.  The author has i n many i n s t a n c e s  n o t i c e d a very l o o s e host c e l l envelope around the Leucocytozoon new  'A  1  gametocyte.  which has been d e s c r i b e d as a probable  s p e c i e s i s a round form  ( i . e . both the gametocyte and the  host c e l l are round), not u n l i k e t h a t of the o l d mature forms of L. bonasae.  In f a c t the s i z e range of the two overlaps  q u i t e c o n s i d e r a b l y . The s t r i k i n g d i f f e r e n c e between the i s the shape of the host n u c l e u s .  In Leucocytozoon  two  'A' the  host nucleus i s t r i a n g u l a r and s i t s on top of the p a r a s i t e l i k e a cap while i n the o l d mature form of L. bonasae the host nucleus i s only a narrow s t r i p o f chromatin on one of the  side  parasite. The o n l y other round Leucocytozoon  t h a t has been  d e s c r i b e d as having a t r i a n g u l a r host nucleus s i t t i n g on i t , i s L. f r i n g i l l i n a r u m Woodcock 1910. between these two  The  species i s i n t h e i r  chief  size  difference  (Leucocytozoon  i s at l e a s t 2-3/U l o n g e r than L. f r i n g i l l i n a r u m ) .  Furthermore  these two are found i n d i f f e r e n t f a m i l i e s of b i r d h o s t . f r i n g i l l i n a r u m i s d e s c r i b e d from the f i n c h e s w h i l e 'A  1  i s from blue grouse.  Attempts  Pearson, and Bennett  L.  Leucocytozoon  by t h i s author to  domestic c h i c k s w i t h L. bonasae have f a i l e d ; and  'A'  infect  Fallis,  (1953) were not a b l e to i n f e c t  ruffed  grouse, c h i c k e n s , t u r k e y s , and pheasants with L. simondi of  -42of ducks. species  These experiments seem to i n d i c a t e that  are q u i t e host s p e c i f i c , and  t h a t L.  fringillinarum  i n f e c t i n g a blue grouse i s h i g h l y improbable. be s a i d t h a t Leucocytozoon  'A',  though i t may  f r i n g i l l i n a r u m i n form, i s a d i s t i n c t  Leucocytozoon  Hence i t  may  resemble L.  species.  Trypanosoma. In the present i n small numbers i n blood grouse out  s t u d i e s , trypanosomes have been found smears of 54 a d u l t and y e a r l i n g blue  of a t o t a l of 62  examined from the three study areas  i n Vancouver I s l a n d d u r i n g the summers of 1963  and 1964.  The  percentage i n f e c t i o n of adult and y e a r l i n g b i r d s i s 84%, i s s l i g h t l y higher (1953  )«  higher  than t h a t r e p o r t e d by Adams and  which  Bendell  As f o r c h i c k s , the percentage i n f e c t i o n i s v e r y much than t h a t r e p o r t e d by previous  from chicks  made at the 1963  workers.  Blood smears  Game Check at Campbell R i v e r  showed a 70% i n f e c t i o n (Table I I ) , whereas Adams and (1953) r e p o r t e d  a 17%  infection i n chicks.  A l l specimens observed from blood the l a r g e S-shaped stage d e s c r i b e d as the most mature.  Bendell  smears were i n  by Novy and MacNeal  (1905)  In a l l , nineteen specimens were measured;  the b a s i s on which the measurements were made are shown i n Figure and oval  2.  The  specimens measure 50.5-55/u  4 . 5 - 6 / 1 (average 5/1 w i d e ) . ( 1 . 5 x l y u ) and  The  (average 53.2/u)  kinetoplast i s s l i g h t l y  l i e s about 12.6^1 from the p o s t e r i o r  I t s t a i n s q u i t e dark r e d w i t h Giemsa S t a i n . undulating  long  membrane s t a i n s pink and  The r i m of  is p l a i n l y v i s i b l e  end. the along  -43= most of the body l e n g t h .  The  r a t h e r l o n g , about 5-7/u. pale r e d . diameter  f r e e f l a g e l l u m appears to be  The nucleus i s rounded and  stairis  I t l i e s on the outer c u r v a t u r e of the body. i t i s approximately  degree of f l a t t e n i n g dark b l u e .  4yu,  In  the s i z e v a r y i n g w i t h the  of the specimen.  The  cytoplasm  stains  A number of specimens show myonemes, the number  of these vary from 5-7«  In some specimens, these myonemes  can be seen r u n n i n g p a r a l l e l t o each other along most o f the l e n g t h of the body. The  l e n g t h s and some o f the other measurements seem  to f i t those of T. g a l l i n a r u m Bruce et a l . 1911  (Table I X ) ,  but as trypanosomes i n g e n e r a l are very polymorphic c h a r a c t e r i s t i c s should a l s o be c o n s i d e r e d i n the Blood from an i n f e c t e d b i r d was  the c u l t u r a l  identification.  c u l t u r e d i n the  d i p h a s i c medium employed by Tobie, von Brand, and Mehlman (1950) f o r the study T. gambiense and T. r h o d e s i e n s e . incubated a t 25 b f o r a p e r i o d o f s i x days. c  twenty-four  A f t e r f o r t y - e i g h t hours i n c u b a t i o n at 25°C scanty  s l e n d e r leptomonad forms were observed.  These forms average  &.&ja l o n g and 1 . 5 y u w i d e ; the f l a g e l l u m i s approximately long.  was  No d i v i d i n g forms  were ever detected i n the c u l t u r e i n the f i r s t hours.  It  Some of the leptomonad forms show a s l i g h t  the a n t e r i o r end;  near  i n s t a i n e d specimens t h i s area seems to  occupied by the nucleus and the k i n e t o p l a s t , both r e d i n Giemsa's S t a i n . s t a i n s pale b l u e .  bulge  7/u  The  cytoplasm  staining  i s vacuolated  and  be  -44-  By the t h i r d day o f i n c u b a t i o n the c u l t u r e was growi n g very w e l l , s t i l l  only leptomonad stages were seen.  the f o u r t h day the vtfiole c u l t u r e was teeming w i t h l o n g leptomonad stages,  On slender  but there were a l s o some shorter and  rounder forms which seemed to be the d i v i d i n g forms. long slender forms had also s t a r t e d t o a g g l u t i n a t e . begins with two c e l l s s t i c k i n g together ends which s l i g h t l y o v e r l a p .  The This  at t h e i r p o s t e r i o r  In the l i v i n g p r e p a r a t i o n s the  l i n e of j u n c t i o n cannot be made out, and as a r e s u l t the double c e l l appears to be a s i n g l e organism with each end.  Also,  ' t r i p l e t s ' were present,  assumed that a t h i r d c e l l may attach end  t o the a g g l u t i n a t e d p a i r .  a whip a t  i n these i t i s  i t s e l f by i t s p o s t e r i o r  F i n a l l y more c e l l s come i n and  j o i n the same way, e v e n t u a l l y g i v i n g r i s e to t a n g l e s of hundreds of c e l l s .  In these groups or masses the whips are  always on the o u t s i d e or the p e r i p h e r y ,  whereas i n r o s e t t e s  as d e s c r i b e d by Novy and MacNeal (1905) the whips are always on the i n s i d e .  On the f i f t h day of i n c u b a t i o n the a g g l u t i n -  a t i o n was more pronounced and by the s i x t h day m u l t i p l i c a t i o n had not ceased as the a g g l u t i n a t e d masses were  bigger.  On the s i x t h day of i n c u b a t i o n , f o u r c u l t u r e tubes (A, B, C, and D)< were incubated  a t 39^  artiile tubes E and F  were l e f t a t 25°C« Twenty-four hours l a t e r , a l l f o u r tubes a t 3#°c had mostly m e t a c y c l i c  trypanosomes and a few c r i t h i d i a l  forrr?; the two c u l t u r e tubes t h a t were l e f t a t 25°C had nothing  but leptomonad stages.  A l l s i x c u l t u r e s were q u i t e  -45healthy as there was  no s i g n o f b a c t e r i a l contamination  and  the leptomonad forms at 2#°C were d i v i d i n g v e r y r a p i d l y . By the e i g h t h , n i n t h and t e n t h day of c u l t u r e the tubes a t 25°C had reached the peak of growth and t h e r e were v i r t u a l l y thousands of leptomonad stages i n a s i n g l e drop of c u l t u r e medium, a l l clumped together i n masses.  Although  no  q u a n t i t a t i v e s t u d i e s were c a r r i e d out, i t seemed t h a t the number of m e t a c y c l i c trypanosomes i n the tubes incubated at 3#°C remained r e l a t i v e l y the same. i s 10yu  The m e t a c y c l i c trypanosome on the average l o n g and  3/u  wide a t i t s widest.  The k i n e t o p l a s t , which  stains  r e d i n Giemsa's S t a i n i s approximately 3• 5/u. from the p o s t e r i o r end.  The nucleus i s near the center of the body.  The  i n g membrane which does not s t a i n i n Giemsa's S t a i n studied i n l i v e preparations. approximately 1.5/u  was  The f r e e f l a g e l l u m measures  i n dead u n s t a i n e d preparations i n s a l i n e .  On the tenth day of c u l t u r e b a c t e r i a l was  undulat-  contamination  seen i n tube A, the leptomonad p o p u l a t i o n d e c l i n e d very  r a p i d l y and twenty-four hours l a t e r t h e r e was l e f t i n the c u l t u r e tube.  v i r t u a l l y none  C u l t u r e tube B (39°C) was  i n o c u l a t e d i n t o a ' c l e a n ' y e a r l i n g blue grouse. i n o c u l a t i o n was made subcutaneously. domestic  Two  The  four-week o l d  c h i c k s were l i k e w i s e i n f e c t e d with the m e t a c y c l i c  forms from two  other c u l t u r e tubes  domestic c h i c k was  (C and D).  A third  i n j e c t e d with a l l the leptomonad forms  from a 25°C c u l t u r e .  -46-  Examination o f the e x p e r i m e n t a l b i r d s f o r the presence of  S-shaped  trypanosomes  i n the c i r c u l a t i n g blood was done  twenty-four hours l a t e r by d i r e c t examination o f b l o o d from the  brachial vein.  A l l b i r d s were n e g a t i v e f o r p a r a s i t e s i n  the  f i r s t twenty-four hours.  S-shaped  trypanosomes were  d e t e c t e d i n the blood of the y e a r l i n g blue grouse f o r t y - e i g h t hours l a t e r by d i r e c t b l o o d examination.  Blood from a l l t h r e e  domestic c h i c k s were s t i l l n e g a t i v e s e v e n t y - f o u r hours a f t e r i n o c u l a t i o n of the c u l t u r a l forms.  This was confirmed by the  absence o f leptomonad forms i n c u l t u r e s made from the b l o o d of t h e domestic c h i c k s obtained f o r t y - e i g h t and n i n e t y - s i x hours a f t e r t h e i n o c u l a t i o n o f the c u l t u r a l forms i n t o the birds. Black f l i e s  d i s s e c t e d f i v e t o s i x days a f t e r they  were c o l l e c t e d from the exposure grouse had thousands of leptomonad forms i n them. gut  These were r e c o v e r e d i n the f o r e  and i n the v i c i n i t y of the s a l i v a r y g l a n d s .  These are i n  a l l p r o b a b i l i t y the d i v i d i n g forms o f the trypanosomes the  exposure grouse.  from  They are not u n l i k e some o f the leptomonad  forms from the i n - v i t r o c u l t u r e study (Table X ) . (1961) had s t a t e d that trypanosomes  Manwell  i n c u l t u r e s assume the  stages normally found i n the i n s e c t v e c t o r s . forms were ever d e t e c t e d i n d i s s e c t e d  No m e t a c y c l i c  flies.  Discus s i o n The measurements of the S-shaped the  trypanosomes  from  blood of the blue grouse seem t o i n d i c a t e t h a t i t is T . g a l l i n a r u m  TABLE  VIII  Summary of Experimental Work on Blue Grouse  C u l t u r e Temperature C u l t u r e Forms Tube Day 0-Day 6 Day 0-Day 6  Temperature C u l t u r e Forms Day 6-Day 10 Day 6-Day 10  Trypanosomes  Bird infected with Day 10 forms  Result of Infection  A  25°C  leptomonads  39°C  metacyclic trypanosome s  B  25°C  leptomonads  39 °C  metacyclic trypanosomes  Yearling Blue Grouse  C  25°C  leptomonads  39°C  metacyclic trypanosomes  4 week o l d Domestic Chick  negative  D  25°Q  leptomonads  39°C  metacyclic trypanosomes  4 week o l d Domestic Chick  negative  E  25°GJ  leptomonads  25°C  leptomonads  4 week o l d Domestic Chick  negative  F  25°C  leptomonads  25°C  leptomonads  S-shaped Trypanosomes i n c i r c u l a t i n g blood  TABLE  IX  Comparison o f Blue Grouse Trypanosomes w i t h measurements o f Trypanosoma avium and T. g a l l i n a r u m as p u b l i s h e d i n l i t e r a t u r e  Trypanosome  T. avium  Author  Total Length  Width  PK distance  Baker  49.6^  5.1yu  12.^u  Wenyon (1910)  T.gallinarum  Bruce e t al(1911)  T.gallinarum  Wenyon  Trypanosoma from Blue Grouse  /TL  C1910)  Woo  X  From Bennett, 1 9 6 1 .  -  -  52.8^u  11.7/1  -  -  53.2/u  5/i  12.6/u  PN distance  P N  /TL  0.26  23.5/1  0.47  0.43  -  0.57  0.22  25/i  0.47  0.22  -  0.51  0.24  26.2/u  0.49  (1956)  X  T. paddae  P K  TABLE  X  Summary o f Measurements and R a t i o s of Blue Grouse Trypanosomes and t h o s e of c u l t u r a l forms  Trypanosome Form  Source o f Specimens  No. measured  Total Length (T.L.)  S-shaped Trypanosome Form Metacyclie Trypanosome Form  Circulating Blood  19  Culture (39-40°C)  15  ICyu  21  8.8/u  25  10.2 / i  Leptomonad - Form  Culture  Leptomonad - Form  Simulium  (25°C)  Width a t Nucleus  P.K. d i stance  P K  /TL  P.N. d i stance  /TL  (W)  Free Flagellum Length  12.6/t  0.24  26.2/1  0.49  5-7/1  3.5/i  0.35  4.6/i  0.46  1.5/i  1.5/a  5.56  0.63  5.56  0.63  7/i  2.24/1  6.16  0.61  6.16  0.61  6.5/i  53 .2Cyu  -50Bruce et a l . 1911  (Table IX), but f a i l u r e  domestic c h i c k s w i t h the m e t a c y c l i c  to i n f e c t young  forms from the  c u l t u r e p o s i t i v e l y negate t h i s i d e n t i f i c a t i o n F a l l i s i n a personal  communication to Dr.  in-vitro  (Table  J.R.  VIII).  Adams f e e l s  that the trypanosomes found i n r u f f e d grouse i n Ontario £• avium.  Bennett (1961) had  also t e n t a t i v e l y i d e n t i f i e d  trypanosomes from v a r i o u s b i r d s i n Algonquin Park, as T. avium.  is the  Ontario,  His l i s t of n a t u r a l l y i n f e c t e d b i r d s i n Algonquin  Park i n c l u d e d r u f f e d grouse.  The  c u l t u r a l c h a r a c t e r i s t i c s of  the trypanosomes from Vancouver I s l a n d blue grouse d i d not resemble those of T. avium as given by Novy and MacNeal Novy and MacNeal (1905) i n d i c a t e d t h a t r o s e t t e s were abundant i n T. avium c u l t u r e s incubated (approximately 2 2 ° C ) .  c u l t u r e s , there were only a g g l u t i n a t i o n s of the  u n t i l more i n f o r m a t i o n  quite  at room temperature  No r o s e t t e s were ever found i n  stages a f t e r the f o u r t h day  (1905).  of i n c u b a t i o n  concerning the l i f e  my  leptomonad  at 25°C. However, c y c l e and  host  s p e c i f i c i t y are a v a i l a b l e , t h i s author h e s i t a t e s t o a s s i g n the trypanosome t o any species  new  c u l t u r e s were incubated  a t 25°C during  the  s i x days because t h a t i s the average summer temperature  i n the f i e l d . at  or to e r e c t a  to c o n t a i n i t . The  first  e x i s t i n g species  At t h i s temperature the  a tremendous r a t e , but  detected.  c u l t u r e s were d i v i d i n g  only leptomonad forms were ever  F i e l d s t u d i e s a l s o i n d i c a t e d that  stages were present  i n the f l i e s  only  leptomonad  (Simulium aureum and  One phi a  minus) f i v e t o s i x days a f t e r they had f e d on the i n f e c t e d grouse.  No leptomonad stages were d e t e c t e d i n C u l i c o i d e s  during the same p e r i o d .  T h i s does not mean that  s p e c i e s are not v e c t o r s f o r t h i s trypanosome.  Culicoides  As the  i n f e c t i o n i n the grouse was very l i g h t , the chance o f a C u l i c o i d e s p i c k i n g up a trypanosome i n the blood meal was a t l e a s t f i v e t o s i x times l e s s than that l e s s than 10% o f the b l a c k f l i e s stages. as  of a b l a c k f l y ; and  d i s s e c t e d had leptomonad  Bennett (1961) r e p o r t e d t h a t any b i t i n g f l y can a c t  v e c t o r f o r T. avium i n Algonquin Park.  With t h i s study i n  mind t h i s author f e e l s t h a t the C u l i c o i d e s spp. are p o t e n t i a l vectors.  F a i l u r e t o f i n d trypanosomes i n the C u l i c o i d e s  d i s s e c t e d t h i s summer may o n l y i n d i c a t e t h a t they a r e not e f f i c i e n t v e c t o r s i n cases of low i n f e c t i o n s i n the host b i r d s . In t h e i n - v i t r o s t u d i e s a l l the leptomonad stages changed t o the m e t a c y c l i c trypanosomal forms i f the  incubation  temperature was r a i s e d t o 3 9°C f o r twenty-four h o u r s : i n those t h a t were l e f t a t 25°C t h e r e were only leptomonad s t a g e s .  The  r a i s i n g o f the i n c u b a t i o n temperature from 25°C t o 39°C would simulate the temperature change a f f e c t i n g the leptomonads i f they were i n o c u l a t e d from the f l y t o t h e b i r d .  Since a l l the  leptomonads changed to the trypanosomal forms at t h i s h i g h e r temperature, i t i s not too presumptious to p o s t u l a t e t h a t the leptomonads can be the i n f e c t i v e stage ( t h a t i s , i t can cause an i n f e c t i o n i f i t were i n o c u l a t e d i n t o a b i r d ) . Experimental s t u d i e s i n d i c a t e t h a t the m o r t a l i t y r a t e o f f l i e s  would  -52-  i n c r e a s e very much i f the temperature was f o r any  l e n g t h of time.  r a i s e d above 30 C  T h i s would t e n d to i n d i c a t e , that  under n a t u r a l c o n d i t i o n s the f a c t o r r e s p o n s i b l e  f o r the  change of the leptomonad stage t o the m e t a c y c l i c  trypanosome  stage i n the f l y ( i f t h i s occurs at a l l ) would be p h y s i o l o g i c a l stimulus stimulus  such as The  i n j e c t i n g the  from the f l y r a t h e r than an  be the  l i f e c y c l e of the  trypanosome was  c u l t u r a l metacyclic  No  forms may  1  been proved  leptomonad forms from the no  can  culture  'clean' grouse  s t a t e d e a r l i e r i n the d i s c u s s i o n the  be the  no m e t a c y c l i c  'clean  forms from the c u l t u r e  were ever i n j e c t e d i n t o grouse as t h e r e was As  completed by  forms i n t o a  Hence i t has  t h a t the m e t a c y c l i c  i n f e c t i v e form.  available.  external  temperature.  y e a r l i n g grouse (Table V I I I ) . experimentally  a  leptomonad  i n f e c t i v e forms i n n a t u r a l t r a n s m i s s i o n  forms were ever found i n the f l i e s  as  dissected.  -57SUMMARY AND I.  CONCLUSIONS  Haemoproteus 1. A new  s p e c i e s of Haemoproteus was  Vancouver  I s l a n d blue grouse  f u l i g i n o s u s Ridgewav).  d e s c r i b e d from the  (Dendragapus  The gametocytes  obscurus  of  H.dendragapi  are round and the occurrence of t h i s s p e c i e s seems to be l o c a l i s e d i n the Nanaimo Lakes A r e a . 2. The r a t e of gametocyte development the Vancouver  of H. c a n a c h i t e s i n  Island blue grouse seems to be much more  r a p i d than t h a t d e s c r i b e d by F a l l i s f o r H, c a n a c h i t e s i n r u f f e d grouse i n O n t a r i o . 3. Young t i s s u e  stages of H. c a n a c h i t e s were d e s c r i b e d  from the lung smears of grouse  chicks.  I I . Leucocytozoon 4. The l i f e  c y c l e of L. bonasae was  completed by a  new  v e c t o r (Cnephia minus); also Simulum aureum as a v e c t o r f o r L. bonasae was  confirmed.  Attempts to i n f e c t  domestic c h i c k s were u n s u c c e s s f u l . 5.  D e s c r i p t i o n s of the change i n form of L. bonasae gametocytes w i t h age was  accomplished by making  s u c c e s s i v e smears from an i n f e c t e d 6. A probable new  bird.  Leucocytozoon s p e c i e s was  the Campbell R i v e r Area, Vancouver  d e s c r i b e d from  Island.  I I I . Trypanosoma 7.  D e s c r i p t i o n of the S-shaped form i n the blood o f the b i r d was  given.  -58= 8.  I n - v i t r o c u l t u r e of the  trypanosome was  i n a d i p h a s i c medium, and  accomplished  d e s c r i p t i o n o f the f l y  stages were compared with those of the c u l t u r a l forms. 9.  The  life  c y c l e of the trypanosome was  i n j e c t i n g the m e t a c y c l i c a  forms from the  ' c l e a n ' y e a r l i n g grouse.  c h i c k s were  completed by culture into  Attempts to i n f e c t domestic  unsuccessful.  T h i s study confirms the work of e a r l i e r workers (Fowle Adams and B e n d e l l 1953 ; and  B e n d e l l 1955)  who  found that  1946; the  Vancouver I s l a n d blue grouse are q u i t e h e a v i l y i n f e c t e d w i t h p a r a s i t i c blood provide present.  protozoa o f three  d i f f e r e n t genera,  and  a p r e c i s e c h a r a c t e r i z a t i o n o f the s p e c i e s which are  -59-  1  P a r a s i t e Length.  2  P a r a s i t e Width.  3  Host C e l l Nucleus Length.  4. *••'•• Host C e l l Nucleus Width. 5  P a r a s i t e - H o s t C e l l Length (from one end o f the a t t e n u a t i o n to the o t h e r . )  6...... P a r a s i t e - H o s t C e l l Width host c e l l complex.)  (' the whole width of the p a r a s i t e \  -60-  1  P o s t e r i o r T i p to  2  P o s t e r i o r T i p t o Center of Nucleus.  3...... Length of Free 4  Kinetoplast.  Flagellum.  Width of P a r a s i t e  ( j u s t a n t e r i o r of  T.L.... T o t a l Length of P a r a s i t e t i p to, the a n t e r i o r end :  starts).  nucleus).  ( l e n g t h from the  posterior  j u s t b e f o r e the f r e e  flagellum  -61-  F i g u r e 4. Photomicrograph o f Immature Male and Female Haemoproteus dendragapi Gametocytes.  -62-  F i g u r e 5 . P h o t o m i c r o g r a p h o f Mature Male dendragapi Gametocytes.  Haemoproteus  -63-  F i g u r e 6 . Photomicrograph o f A t t e n u a t e d Form (Type I ) of Leucocytozoon  bonasae.  F i g u r e 7. Photomicrograph o f A t t e n u a t e d Form (Type I I ) o f Leucocytozoon  bonasae.  -64-  F i g u r e 8 . Photomicrograph o f Round Female Leucocytozoon bonasae  Gametocyte.  F i g u r e 9. Photomicrograph o f Female Leucocytozoon Gametocyte.  T  A  -65-  F i g u r e 10. Photomicrograph of T i s s u e Stages o f Haemoproteus canachites.  F i g u r e 12. Photograph of F i e l d Equipments f o r the C o l l e c t i o n and Maintenance o f Engorged  Flies.  -67-  F i g u r e s 13, 14, 15, and 16 a r e p r e p a r e d f r o m f i x e d and s t a i n e d p r e p a r a t i o n s . A l l f i g u r e s a r e drawn t o same s c a l e .  F i g u r e 13. E x f l a g e l l a t i o n o f microgametocyte o f L. bonasae i n stomach c o n t e n t s o f S i m u l i u m aureum t h a t i n g e s t e d gametocytes 1 hour p r e v i o u s l y .  F i g u r e 14. Zygote of L. bonasae i n stomach c o n t e n t s o f Gnephia minus t h a t i n g e s t e d gametocytes 12 hours p r e v i o u s l y .  F i g u r e 15. O o k i n e t e o f L. bonasae i n stomach c o n t e n t s o f Cnephia minus t h a t i n g e s t e d gametocytes 12 hours p r e v i o u s l y .  F i g u r e 16. S p o r o z o i t e o f L. bonasae r e c o v e r e d from s a l i v a r y g l a n d o f Cnephia minus 10 days a f t e r i n g e s t i n g b l o o d meal.  Figure  Figure  16,  15.  -68. LITERATURE CITED Adams,J.R. and B e n d e l l , J.F. 1953. A h i g h i n c i d e n c e of blood p a r a s i t e s i n a p o p u l a t i o n of sooty grouse. J . P a r a s i t . 39 Sect.2 ( S u p p . ) l l . Anderson,R.C. 1956. The l i f e c y c l e and seasonal t r a n s m i s s i o n of Q r n i t h o f i l a r i a f a l l i s e n s i s Anderson, a p a r a s i t e of domestic and w i l d ducks. (Jan. J . Zool. 34: 485-525. A t c h l e y , F.O.  1951. Leucocytozoon andrewsi n. sp. from chickens observed i n a survey of domestic animals i n South C a r o l i n a . J . P a r a s i t . 3 7 : 4 8 3 - 4 8 8 . 1 9 5 6 a . S t u d i e s on Trypanosoma avium. I. Incidence i n some b i r d s of H e r t f o r d s h i r e . Parasitology  Baker, J.R.  46:308-320.  , 1956b. S t u d i e s on Trypanosoma avium. I I . T r a n s m i s s i o n by Ornithomyia a v i c u l a r i a . P a r a s i t o l o g y 46:. 3 2 1 - 3 3 4 . B e n d e l l , J.F.  1955. Disease as a c o n t r o l of a p o p u l a t i o n of blue grouse, Dendragapus obscurus f u l i g i n o s u s (Ridgeway) Can. J . Z o o l . 3 3 : 195-223.  Bennett, G„F,  i960. On some o r n i t h o p h i l i c b l o o d - s u c k i n g D i p t e r a i n Algonquin Park, O n t a r i o , Canada. Can. J . Z o o l . 3 8 : 377=389. 196l. On the s p e c i f i c i t y and t r a n s m i s s i o n o f some a v i a n trypanosomes. Can. J . Z o o l . 39:17=33.  , and F a l l i s , A.M. i 9 6 0 . Blood p a r a s i t e s of b i r d s i n Algonquin Park, O n t a r i o , and a d i s c u s s i o n o f t h e i r t r a n s m i s s i o n . Can. J . Z o o l . 38:261=273. 1935» Blood p a r a s i t e s of r u f f e d grouse (Bonasa-umbellus) and spruce grouse (Canachites canadensis),, w i t h d e s c r i p t i o n of Leucocytozoon bonasae n.sp. Can. J . Research, 12: 646-650.  C l a r k e , C.H.D.  Coatney, G.R.  . 1936. A check l i s t and host-index of the Genus Haemoproteus „ J . P a r a s i t . 2 2 : 8 8 - 1 0 5 . , 1937. A c a t a l o g and h o s t - i n d e x of the Genus Leucocvtozobn. J . P a r a s i t . 2 3 : 2 0 2 - 2 1 2 ,  -69-  1955* The development of megalosehizonts of Leucocytozoon simondi Mathis and Leger. J . P r o t o z o o l . 2: 1 5 3 - 1 6 7 .  Cowan, A.B.  , and P e t e r l e , T . J . 1957. Leucoytozoon bonasae Clarke i n Michigan s h a r p - t a i l e d grouse, J . W i l d l i f e Management 21: 4 6 9 - 4 7 1 .  .  Diamond, L.S. and Herman, C M . 1954. Incidence of trypanosomes i n Canada goose as r e v e a l e d by bone marrow c u l t u r e . J . P a r s i t . 40: 1 9 5 - 2 0 2 .  i  Dorney, R .S. and Todd, A C . i960. Spring incidence of r u f f e d grouse b l o o d p a r a s i t e s . J . P a r a s i t . 4 6 : 6 8 7 - 6 9 4 . i!  0  E r i c k s o n , A.B.  1953. Leucocytozoon bonasae i n r u f f e d grouse; i t s p o s s i b l e r e l a t i o n s h i p to f l u c t u a t i o n s i n numbers of grouse. J . W i l d l i f e Management  17:  536-538.  1945. P o p u l a t i o n trends and blood p a r a s i t e s i n r u f f e d grouse i n O n t a r i o . J . W i l d l i f e Management 9.: 203=206.  F a l l i s , A.M.  .5  1946. Plasmodium circumflexum i n r u f f e d grouse in Ontario. J . P a r s i t . 32: 345-353. Anderson, K.C., and Bennett. 1956. Further Observations on the t r a n s m i s s i o n and development of Leucocytozoon simondi. Can.J. Z o o l . 3 4 : 389-404< and Bennett, G.F. 1958. T r a n s m i s s i o n of Leucocytozoon bonasae C l a r k e t o r u f f e d grouse (Bonasae umbellus) by the b l a c k f l i e s Simulium l a t i p e s MG. and Simulium aureum F r i e s . Can. J . Z o o l . 3 6 : 533=539. i960. D e s c r i p t i o n of Haemoproteus c a n a c h i t e s n. sp. (Sporozoa: Haemoproteidae) and sporogony i n C u l i c o i d e s (DipterasCeratopogonidae). Can. J . Z o o l . 3 8 : 4 5 5 - 4 6 4 . 1961. Sporogony of Leucocytozoon and Haemoproteus i n S i m u l i d s and Ceratopogonids and a r e v i s e d c l a s s i f i c a t i o n of the Haemosporidina Can. J . Z o o l . 3 9 : 2 1 5 - 2 2 8 . . , 1962. Observations on the sporogony o f Leucocytozoon mirandae., L. bonasae. and L. f r i n g i l l i n a r u m (Sporozoa:LeucocytozoidaeJ. Can. J . Z o o l . 4 0 : 395=400.  -70Pearson, J.C., and Bennett, G.F. 1954. On the s p e c i f i c i t y of Leucocytozoon. Can. J . Z o o l . 3 2 : 120-124.  F a l l i s , A.M.,  T  Fantham, H.B.  1910. Observations on th e p a r a s i t i c protozoa of the r e d grouse (Lagopus s c o t i c u s ) w i t h a note on the grouse f l y . Proc. Z o o l . Sbc. London 2:692-708. 1946. The b l o o d p a r a s i t e s of blue grouse. Science 103• 7 0 8 - 7 0 9 .  Fowle, C D .  1944. The blood p r o t o z o a of North American b i r d s . B i r d Banding 15; 8 9 - 1 1 2 .  Herman, C M .  1932. Am.  Huff, C.G.. .  and Wood, D.M. 1957. B i t i n g midges ( D i p t e r a : Ceratopogonidae) as i n t e r m e d i a t e hosts for Haemoproteus of ducks. Can. J . Z o o l . 35:425-435*  S t u d i e s of Haemoproteus o f mourning doves. J . Hyg. 16: 618-623.  , 1942. Schizogony and gametocyte development i n Leucocytozoon simondi and comparisons w i t h Plasmodium and Haemoproteus. J . I n f e c t . D i s e a s e 71: 1 8 - 3 2 . 7  I960.  Kudo, R.R.  Manwell, R.D. Novy, F.C.  C h a r l e s C. Thomas (4th E d i t i o n )  Protozoology.  1961. I n t r o d u c t i o n to P r o t o z o o l o g y . S t . M a r t i n ' s P r e s s . New York.  and MacNeal, W.J. 1905. On the trypanosomes of b i r d s . J . I n f e c t . Disease, 2 : 2 5 6 - 3 0 8 .  O'Roke, E . C  1934. A m a l a r i a - l i k e d i s e a s e of ducks. Univ. of Michigan School of F o r e s t r y and C o n s e r v a t i o n B u l l . No.4.  .Sambon, L.W.  1908. Remarks on a v i a n Haemoprotozoa of the Genus Leucocytozoon Danilewsky. J . Trop. Med. f  and Hyg. 1 1 : 3 2 5 - 3 2 8 .  S h o r t t , H.E.  and Cooper, W. 1948. S t a i n i n g of microscopical s e c t i o n s c o n t a i n i n g p r o t o z o a l p a r a s i t e s by m o d i f i c a t i o n o f McNamara's method. Trans. Roy. Soc. Trop. Med. and Hyg. 4 1 : 427.  Shute, P. and Maryon, M. 1961. Study of M a l a r i a .  Laboratory Technique f o r the J.A. C h u r c h i l l , London. p p : 1 7 - 2 1 .  -71Tobie, E., von Brand, T., and Mehlman, B. 1950. Cultural and p h y s i o l o g i c a l o b s e r v a t i o n s on Trypanosoma rhodesiense and T. gambiense . J . Par as i t . 3 6 :4#. Wenyon, C M .  1926.  Protozoology.  London and B a l t i m o r e .  Wetmore, P.W . 1936. A s p e c i e s of Plasmodium from the t a i l e d grouse i n f e c t i v e t o other b i r d s . W i l d l i f e Manag. 3:361-365. Woodcock, H.M.  sharpJ.  1910. S t u d i e s on a v i a n haemoprotozoa. I. On c e r t a i n p a r a s i t e s o f the c h a f f i n c h ( F r i n g L l l a coelebs) and the r e d - p o l l ( L i n o t a rufescens). Quart. J . M i c r o s c o p . S c i .55:.641-740.  

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