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Epizootiological factors in three outbreaks of pseudotuberculosis in British Columbia canaries Stovell, Peter Lawrence 1963

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EPIZOOTIOLOGICAL FACTORS IN THREE OUTBREAKS OF PSEUDOTUBERCULOSIS IN BRITISH COLUMBIA CANARIES by PETER LAWRENCE STOVELL D.V.M. U n i v e r s i t y of Toronto 1952 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE ' OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1963 I n presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University ©f B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission,, ABSTRACT Three naturally-occurring epizootics of Paeteurella  pseudotuberculosis in canaries were studied. Cultural details and gross histopathological lesions were described for birds from two of the aviaries. Epizootlological observations were made in a l l three cases following v i s i t s to the premises and recovery of data on management, f i r s t c l i n i c a l signs, and mortalities. A reasonably complete study was made of early and current literature concerning pseudotuberculosis infections and incidence in birds and mammals, both feral and domesticated. Although this disease has been commonly reported in canaries in Europe from 186k- onwards, the epizootics herein reported are, as far as the author is aware, the f i r s t bacterlologically con-firmed canary Infections to be reported on the North American Continent. Because of the high mortalities encountered, and because of the known potential of the causative organism to produce human disease, the epizootlological considerations were extended to Include experimental studies on the faecal excretion rate of viable Pasteurella pseudotuberculosis by naturally and a r t i f i c i a l l y Infected canaries. W i t h the e v o l u t i o n o f s u i t a b l e c u l t u r i n g t echniques , the r ecovery of v i a b l e organisms of the i n o c u l a t i o n s t r a i n from e x p e r i m e n t a l l y i n f e c t e d canar ie s was performed w i t h ease. Th i s a l l o w e d counts to be c a r r i e d out on t o t a l d a i l y f a e c a l samples from twenty i n o c u l a t e d and f o u r c o n t r o l b i r d s f o r a three-week p e r i o d f o l l o w i n g o r a l i n o c u l a t i o n . I t was found tha t i n the group o f twenty i n o c u l a t e d b i r d s which s u f f e r e d f i v e (20%) m o r t a l i t i e s , some s i x t e e n (20$) , i n c l u d i n g those b i r d s tha t d i e d , excre ted organisms f o r p e r i o d s r ang ing from three up to n ine teen days . Peak ampl i tudes f o r the e s t imated f a e c a l counts of P a s t e u r e l l a p seudotubercu los i s per day v a r i e d from Log U-.3 up to Log 2 . 1 . The f o u r dead b i r d s showed gross l e s i o n s t y p i c a l of n a t u r a l l y - o c c u r r i n g p s e u d o t u b e r c u l o s i s , and y i e l d e d recovery c u l t u r e s from v a r i o u s organs . The remain ing twelve shedder b i r d s a l l . ceased shedding v i a b l e P ^ p seudotubercu lo s i s by the t w e n t i e t h day f o l l o w i n g -the c o n c l u s i o n o f o r a l I n o c u l a t i o n s ( 2 ^ t h e x p e r i -mental d a y ) , and a l though none y i e l d e d a p o s i t i v e c u l t u r e from v a r i o u s organs , there wwre s l i g h t - t o - m a r k e d s p l e n i c l e s i o n s i n a l l but two when they were au tops ied on the t w e n t y - f i f t h e x p e r i -mental day. Four b i r d s which never shed d e t e c t a b l e numbers of v i a b l e P_j_ p seudotubercu lo s i s were found to have no v i s i b l e gross l e s i o n s when s a c r i f i c e d and a u t o p s i e d . Four n o n - i n o c u l a t e d c o n t r o l b i r d s a l s o f a i l e d to shed d e t e c t a b l e numbers of v i a b l e P . p s e u d o t u b e r c u l o s i s . i i i Attempts were made i n the laboratory to al low na tura l ly and experimentally infected canaries to transmit the in fec t ion to healthy contact b i r d s . These attempts were unsuccessful, and i t was concluded from t h i s and from d i r ec t observations on the na tura l epizoot ics that predisposing factors other than the presence of the organism (such a s . c l i m a t i c or poor-management s t ress , or g a s t r o - i n t e s t l n a l i r r i t a t i o n ) are required at times for the disease to become epizoot ic i n canaries. From the faecal excret ion rates of v i ab le pseudotuberouloeis measured i n experimentally infected b i rds , and from e p i z o o t i o l o g i c a l obser-va t ions , i t was concluded that canaries in fec t each other ( rather than the in fec t ion coming from a common source) and that they are po ten t i a l spreaders of in fec t ion (or of the in fec t ing organism) to other species i n contact, inc lud ing man. Gross and h is topa thologica l observations of experimentally Infected b i rds corre la ted wi th t he i r faecal counts, and gross pa thologica l observations on na tura l ly- Infec ted b i rds , indica te that les ions i n the bowel w a l l , i n p a r t i c u l a r caecal abscesses, are the les ions which when present predispose to a high po ten t i a l of i n f e c t i v i t y i n the shed faeces. X ACKNOWLEDGMENTS The diagnost ic studies which formed the basis for th i s thesis were part of the service and research work on avian and mammalian diseases which i s ca r r ied out i n B r i t i s h Columbia by the Canada Department of Agr i cu l t u r e , Animal Pathology Laboratories D i v i s i o n of the Health of Animals Branch. My grat i tude i s extended to Dr. P . J . G . Plummer, Di rec to r of Laboratories , s i tuated at the Animal Diseases Research In s t i t u t e i n H u l l , Quebec, for his in te res t and suggestions which made i t possible fo r pseudotuberculosis to be incorporated in to a research project , No. 515, at the Vancouver Laboratory. I am deeply Indebted to Drs. E . V . Langford and J . C . Bankier of the B r i t i s h Columbia Department of Agr i cu l t u r e , Animal Pathology Laborator ies , Vancouver. The former con-t r ibu ted valuable reference data of h i s own work on pseudo-tuberculosis and by h is prompt diagnosis of th i s condi t ion i n Aviary No. and h is generous arrangements allowed me unl imited access to t h i s aviary and use .of a l l data and materials that were derived therefrom. Dr. John Bankier ass i s ted me wi th valuable suggestions for the work and provided cul tures and data on the incidence of pseudotuberculosis i n beaver and ch in -c h i l l a s i n B r i t i s h Columbia. x i As the work progressed, I became the rec ip ien t of much generously given time, suggestions, d i r e c t i o n , and c r i t i c i s m from Dr. James R. Adams of the Department of Zoology, and Dr. Harold E. Taylor of the Department of Pathology.;. Univers i ty of B r i t i s h Columbia. I am grateful to Dr. Ian MacTaggart Cowan, Head of the Department of Zoology, for the i n i t i a l stimulus and consistent encouragement which made th is work poss ib le . When the volume of work increased i n the laboratory phases, I received technica l assistance of outstanding ca l i b r e from Miss B.M. Coles, i n making and checking many hundreds of i so l a t i ons from t issues and faecal samples, the l a t t e r under great pressure; from.Mrs. M.E. Bo iv in and Mrs. L . Watkins i n cu l t u r i ng and preparation of Immaculate glassware and other supplies; from Miss Dale Young for meticulous folloxtf-up b a c t e r i o l o g i c a l work invo lv ing the more sophis t ica ted t es t s , and from Mr. J . Kent Simpson i n preparing excel lent and we l l -o r i en ted t i ssue sections from mater ia l which was i n small and often b r i t t l e por t ions . Dr. Werner Knapp of the In s t i t u t e of Hygiene, Tubingen Un ive r s i t y , Germany has my thanks for typing the B r i t i s h Columbia cul tures and provid ing type cul tures for comparative s tudies . x i i I am indebted t o Mr. J . Schoenfeld, Dr. Alec S t o v r i d e s , and Mr. Kurt Weissenborn f o r valuable t r a n s -l a t i o n s from German papers and to Mr. A. Goldbeck f o r manufacture of the canary b a t t e r y . I wish to thank my former s u p e r v i s o r , Dr. Rob-e r t J . Avery f o r h i s i n t e r e s t and encouragement i n i t i a l l y ; Drs. P.. A. Simon and A. C. M a c N e i l l , my present col l e a g u e s , f o r t h e i r patience and a s s i s t a n c e i n the f i n a l phases of the work, and Mrs. Joyce McCart, Mrs. Colleen Haskins and Mrs. S h i r l e y Walton f o r a s s i s t i n g i n the p r e p a r a t i o n of the t a b l e s and t y p i n g of the manuscript. F i n a l l y , I thank ray w i f e Mary, not only f o r her encouragement and/endurance, but f o r numerous occasions when the evening t e c h n i c a l d u t i e s , or l i t e r a t u r e survey, demanded unexpected assis.tance. i v TABLE OP CONTENTS page GENERAL INTRODUCTION, REVIEW OF THE LITERATURE CONCERNING PSEUDOTUBERCULOSIS IN BIRDS..... ... 5 Canaries and Other Cage Birds................................... 6 F r e e - l i v i n g or Free-ranging Birds..................................... 15 Domesticated Fowl Used for' Human Food............................. 20 DESCRIPTIONS OF THREE EPIZOOTICS OF PSEUDOTUBERCULOSIS IN BRITISH COLUMBIA CANARIES ...... 25 H i s t o r y and Recorded Observations.................................. 25 A v i a r y No. 1, Vancouver, .B.C...,.................................. 25 A v i a r y No. 2, Vancouver I s l a n d (E. Coast) B.C........... 33 A v i a r y No. -3, North Burnaby, B.C........................ 38 Summary of E p i z o o t i o l o g l c a l Considerations f o r the Three A f f e c t e d Aviaries................................................... 4-6 Laboratory Diagnosis of Pseudotuberculosis..................... 47 B a c t e r i o l o g i c a l Diagnosis................................................... 47 P a t h o g e n i c i t y f o r Experimental Animals........................ 52 Pathology Diagnosis......................................... 53 EXPERIMENTAL STUDIES ON THE TRANSMISSION, FAECAL EXCRETION RATE AND PATHOLOGICAL FINDINGS OF PSEUDOTUBERCULOSIS IN CANARIES... ..... ..... . ......... 58 M a t e r i a l s and Methods General........... 59 Execution of Experim.ents.;x. v... ...... .• ... 69 V page Experiment A. ©n Canaries Transferred to the Laboratory from A v i a r y N.©. 3..... 69 Experiment B. Contact I n f e c t i o n w i t h Healthy and A r t i f i c i a l l y I n f e c t e d Birds.................................. 79 Experiment C. Experiment w i t h Q u a n t i t a t i v e Deter-minations ©f Faec a l Dissemination..................... 8^  DISCUSSION AND SUMMARY OF EPIZOOTIOLOGICAL AND EXPERIMENTAL INFECTION STUDIES. ............................................. 129 BIBLIOGRAPHY...... ................. .................. ................... . . 138 APPENDIX.. . . 1^ 7 V i LIST OP TABLES page Table I His tory and epizootiology of Pasteurel la  pseudotuberculosis outbreaks In canaries as reported by various a u t h o r s . . . . . . . . . . . 8" Table I I C l i n i c a l observations and pathological l e -sions noted by various authors i n canaries spontaneously infected with Pas teure l la  t > s e u d o t u b e r c u l o s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Table I I I C l i n i c a l observations and pathologica l les ions noted by the author i n canaries from spontaneous epizoot ics of Pas teure l la  pseudotuberculosis i n fec t ion i n B r i t i s h Columbia. . • 11 Table IV P a r t i a l f indings of various authors r e l a t i n g to spontaneous Pasteurel la pseudotuberculosis , . i n f ec t i on i n pigeons and d o v e s . . . . . . . . . . . . . . . . . . . . . . 16 Table V Pseudotuberculosis i n various w i l d b i r d s . p a r t i a l data of several a u t h o r s . . . . . . . . . . . . . . . . . . . . . 19 Table VI P a r t i a l l i s t of ava i l ab le references In the l i t e r a t u r e concerning.pseudotuberculosis i n c h i c k e n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21-22 Table VTI P a r t i a l l i s t of ava i l ab le references i n the l i te ra ture .concern ing , pseudotuberculosis i n turkeys. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23-24-Table VI I I Record of na tura l losses , b i rds destroyed for autopsy, and post mortem resu l t s for the ep izoot ic of Pas teure l la pseudotuber- culos i s i n Aviary number 1.......................... 31 Table IX Record of mor ta l i t i e s occurring i n No. 3 epizoot ic of spontaneous Pas teure l la pseudotuberculosis in fec t ion i n canar ies . . 44 Table X Standard conversion formula employed for computing t o t a l equivalent v iab le faecal count of Pas teure l la ps eudotuberculos i s per t o t a l twenty-four-hour faecal sample of exper i -mentally infected c a n a r i e s . . . . . . . . . . . . . . . . . . . . . . . . . . 66 v i i page Table XI Record of m o r t a l i t i e s , pa thological l e s ions , and Iso la t ions of Pas teure l la  pseudotuberculosIs from canaries t rans-ferred to laboratory from an aviary during the course of an epizoot ic (Group I t r ans fe r red .30 days .af ter . losses c o m m e n c e d ) . • • « . . . . . . . « » « . 7^ * Table X I I Record of m o r t a l i t i e s , pa thologica l l e s i o n s , and i s o l a t i o n s of Pasteurel la  pseudotuberculosis from canaries t ransferred to the laboratory from an aviary during the course of an e p i -zoo t ic (Group I I t ransferred: 14- days af ter group I , i . e . 44 days af ter losses had c o m m e n c e d ) . . . . . . . . . . . . . . . . . . . . . . . . . . • • 75 Table X I I I Effect of inocu la t ion of broth cul ture o r a l l y i n s i x canaries - s t r a i n "Long-worth P n . Inoculum consisted of 0 . 2 ml Brown's #10 equivalent resuspenslon of twenty-four-hour growth at 3 7 ° C Table XIV Aseptic Technique for c o l l e c t i o n . o f 24--hour faecal samples from c a n a r i e s . . . . . . . . . . . . . . . . . . . . . 93---)94-Table XV Processing and-cu l tu ra l procedures used i n obta ining faecal counts and faecal, dry matter output on a d a i l y b a s i s . . . . . . . . . . . . . . . . . . . . . 95-9*5 Table XVI Grouping of .twenty-four experimental birds according to faecal dissemination d a t a . . . . . . . . . . . . 105 Table XVII Data summary sheet for twenty inoculated b i rds (Experiment C) 10o Table XVIII Conjectural pathways of transmission of Pi 1 pseudotuberculosis associated with ep izoot ic and sporadic I n f e c t i o n . . . . . . . . . . . . . . . . . 132 LIST OP FIGURES page Figure 1 Aviary Mo. 1. Southerly aspect with entrance to service passageway,,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Figure 2 Aviarv No. 1. F l i g h t used for c ana r i e s . . . . . . . . . . . 29 Figure 3 Aviarv No. 1. West F l i g h t housing budgerigars . . 29 Figure b Aviary No. 1. Some in te rna l organs of infected canaries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3° Figure 5 Aviary No. 2. General aspect of aviary.,...., 3* Figure 6 Aviarv No. 2. Close-up view of f l i g h t c c a g e , . . . . . 3^ Figure 7 Aviarv No. 3. Reconstructed l i n e drawing of bu i l d ing housing tiCanaries and other cage bi rds . . . lj-0 - 4/1 Figures Aviary No. 1. Spleen from spontaneous case of 8 & 9 canary pseudotuberculosis. Photomicrographs of necro t ic f o c i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Figures Aviarv No. 3., L i v e r from spontaneous case of 10 $ 11 canary pseudotuberculosis. Photomicrographs of necro t ic areas,!,,.......,..................... . . . . . . . . . 56 .Figures Experiment A. Views of f l i g h t cage.containing 12 & 13 canaries of Group I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Figure 1^  Experiment A. Canary from Group I . Spleen and l i v e r les ions at autopsy (spontaneous Infection) 7° Figure 15 Experiment A f Canary from Group I I . Breast muscle and., l i v e r les ions at.autopsy (spontaneous i n f e c t i o n ) , . ; . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Figure 1.6 Experiment B . Gross view at autopsy of thymus l e s i o n i n canary which died 3^ days fo l lowing o r a l inocu la t ion with P. pseudotuberculosis . . . . . . 23 Figures Experiment C. Views of the canary battery uni t 1 7 - 2 0 which was used for quant i ta t ive faeca l dissemin-a t i on exper iments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 - 27 Figures Graph charts of data co l lec ted d a i l y on faecal 21 - 4-0 counts of v iab le P*. pseudotuberculosis and faecal output i n terms of dry matter . . . . . . . . . . . . . . . . 107-111 Vix;.* page F i g u r e 4:1 E x c r e t i o n of v i a b l e P. pseudotuberculosis by in o c u l a t e d b i r d s , "^otals f o r twenty e x p e r i -mental days. ......................................... 112 F i g u r e 4-2 D a i l y record showing numbers of b i r d s shedding (Group A - Table X V I ) i l l u s t r a t e d by a graph chart......................................... •••••••« 114-F i g u r e 4-3 D a i l y record showing numbers of b i r d s reaching the p o i n t of "recovery" i n terms of ceasing to shed v i a b l e P, pseudotuberculosis (Group A -Table X V I ) . . . . . . . . . . . . . . 115 F i g u r e 44 Experiment CV B i r d No. 1$.. Autopsy showing gross views of spleen.............................. 117 F i g u r e 4-5 Experiment C. Close-up of spleen, p i c t u r e d i n Figure 44......... 117 F i g u r e 4-6 Experiment C. B i r d No. 16 w i t h l e s i o n i n thymus clI*@Q. of* HCClC» 0-9 •'•*•••••• • • • • • •• • • • • • • • • • • XX8> F i g u r e 4-7 Experiment C. B i r d No. 16 showing close-up of swollen and abscessed c a e c a l buds...................... 112 F i g u r e 4-8 Experiment C. B i r d No. 17. Autopsy showing l e s i o n s i n spleen and breast muscles............. 119 F i g u r e s Experiment C. Experimental pseudotuberculosis, 4-9 & 50 canary Nb. 1.6. Photomicrographs showing the histopathology of the caecal abscess............. 121 F i g u r e s Experiment C. Experimental pseudotuberculosis, 51 & 52 canary No. 16. Photomicrographs showing g i a n t c e l l s and bacte r i o p h a g i a i n the cae c a l abscess.. 122 F i g u r e s Experiment C. Experimental pseudotuberculosis, 53 & 54- canary No. 16. Photomicrographs i l l u s t r a t i n g . . the h i s t o p a t h o l o g i c a l changes i n the spleen....... 123 Figures Experiment C. Photomicrographs of spleen 55 & 56 i l l u s t r a t e d i n Figures 53 and 54-» Twort's S t a i n has been used to. show the.presence of b a c t e r i a i n the l e s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124-GENERAL INTRODUCTION Specific baoterlal disease due to Infection with Pasteurella pseudotuberculosis has been known since 122} when i t was f i r s t isolated in the guinea pig by Malassez and Vignal. Subsequently reported isolations indicate that this pathogen is world-wide in i t s distribution, and has a wide host spectrum, (Meyer 1952) (Stafseth 1959) (Burrows 1963). The principal reservoirs appear to be wild rodents and birds, (Meyer 1952) (Wilson and Miles 1955). I n many cases the organism causes progressive disease characterized by pseudotubercle formation and high mortalities in the population involved. pseudotuberculosis infections have economic importance in some domestic animals e.g. sheep,, mink and chinchillas; in birds, e.g. chickens, turkeys and pheasants; and in laboratory animals, in particular the guinea pig, (See Literature Review and Appendix). Only a few generalized fatal cases of pseudo- tuberculosis infeotion were reported in man until i t was dis-covered that tills organism i s the cause of a non-fatal mesenteric lymphadenitis. This condition is common in Europe, and i t has been recognized in California (Goldman 1957) and Alberta (Hnatko and Rodin 1962). In the last fifteen years of the nineteenth 2 century, epizootics due to this infection were reported fre-quently in canaries in Europe. Though recent reports are lacking, infections in canaries may s t i l l be common in Europe; however, the cases herein presented appear to be the f i r s t con-firmed reports for North America to be published or cited. This study was occasioned by the appearance of canary pseudotuberculosis in epizootic form involving three well-separated aviaries in 1952. Two ocourred on the B r i t i s h Columbia lower mainland, and one on Vancouver Island. Preliminary attempts to isolate the causative organism from the faecal material of Infected canaries were unsuccessful. In view of the opinion of ( a t least one authority on the oral route of transmission (Meyer 195*$) > a*14 in view of the high mortality noted in canaries, this seemed surprising. With more study on techniques, isolates were secured with ease from droppings of oanaries a r t i f i c i a l l y infected by the oral route. This allowed quantitative determinations to be made on the basis of plate counts of diluted faecal material. Since completing the above determinations, a human case of mesenteric lymphadenitis in Britain has been linked with the presence of P^ pseudotuberculosis in the faeces of a canary owned by the patient (Daniels 196l). Early in 1963 a confirmed outbreak of canary pseudotuberculosis oocurred in a Vancouver aviary following the introduction of a male breeding bird which was imported from Europe. In July, 1962, a report was submitted from Maryland (Clark and Locke 1962) describing a major epizootic affecting common grackles in an i c t e r i d roost with a normal capacity of about one million birds. Recently an epizootic of guinea pig pseudotuberculosis occurred on a farm in the Fraser River delta area of British Columbia (Stovell 1963). These ourrent incidents help to illustrate the widespread occur-rence and varied forms of disease caused by pseudotuberculosis. Since i t has been established (Knapp 1954-, 1952) that the organism under study causes the abscess-forming reticulocytic lymphadenitis of the mesentery (ARLM) described by Masshoff and Dfllle (1953)* t n © matter becomes of great clinico-pathological significance in human medicine. Mair et a l . (i960) quoted the figures of Aird (19*1-5) for the Royal Hospital for Sick Children, Edinburgh, and their own figures for the Royal Infirmary, Leioester, also involving children. These figures show that of 120 c l i n i c a l l y suspected appendicitis cases in Edinburgh (19^4) and 93 s U Q B cases at Leioester (part of 1959), some thirty-seven (30.2$) and twenty (21.5$) respectively proved to be suffering from "non specifio" or "acute" mesenteric adenitis. While in retrospect l t can be noted that many possible causes have been suggested (but not proven) such cases occurring in the future must, in the light of new knowledge, be considered for differ-ential laboratory diagnosis which includes pseudotuberculosis. 4-From time to time i t i s to be expeoted that natural epizootics in peak-cycle populations of wild rodents and birds w i l l be reflected by periodic increases in the human case rate. There appears to be l i t t l e chance of an improvement being made in this uncontrolled situation, unless the eplzootiologist and epidemiologist Join forces to establish the important connecting links between man and the creatures of his environment. 5 REVIEW OF THE LITERATURE CONCERNING PSEUDOTUBERCULOSIS IN BIRDS 1 Because of the apparent absence of North American reports on pseudotuberculosis in canaries, and in fact the rarity of such reports on other birds as well, a preliminary search of the literature was made. It was noticed that Stafseth (in Beister and Schwarte 1959) gave an excellent description of the disease which was based entirely upon European work except for Beaudette's (19^0) contribution on the blackbird case in New Jersey in 19*39. Recently, Clark and Locke (1962) in des-cribing the major epizootic in grackles, have given no other specific references to pseudotuberculosis in North American birds. It became apparent that there was a dearth of literature reports on pseudotuberculosis in any North American animals or birds, with the probable exception of guinea pigs (which would presumably show in laboratory annual reports) and possibly chinchillas. One large question remains. Are diagnoses in birds and mammals being made but not reported or i s there in fact less pseudotuberculosis in North America than in Europe? 1 For pseudotuberculosis in mammals and man, see Appendix. 6 Various references contained herein appear for the f i r s t time. These references involve canaries, finches, chinchillas, cattle (in BC), chickens, and beaver (in Alberta). In order to better understand the probable factors involved in answering this question, i t was necessary for the disease reports to be studied in detail, covering as many species as possible so that a true picture of epizootlological possibilities could be obtained. The following pages carry tabular and descriptive details of pseudotuberculosis, Reports noted in the literature. Canaries and Other Cage Birds Canaries The f i r s t report of Pseudotuberculosis in canaries (Zurn 1224-) was followed by at least fifteen others. France, Germany, and other European countries including Scandinavia were affected. Most reports appeared in the next thirty years up to 191^-. Possibly the dearth of reports thereafter was due to the condition being considered too commonplace to report In the literature". The presence of canary isolates in l i s t s of strains studied by various workers over the intervening years suggests that the Incidence of infection continues. (Beck and Huck 1 9 2 5 ; Lerche 1 9 2 7 J Pallaske 1 9 3 3 ; Thai 1 9 5 ^ ; Keymer 1 9 5 9 ) . Only one probable case was reported previously in North America, and this 7 was not confirmed bacteriologically. This incident occurred in Pennsylvania in birds recently brought in from Washington, D.C. (Kinyoun 1906). Three outbreaks occurred in British Columbia in one year (Stovell 1959 — herein reported in detail). A fourth outbreak occurred in 1963 in Vancouver, following intro-duction to the aviary of an imported European bird, (Stovell 1963). In view of imports of canaries from Europe especially, and the presence of infection in other species, i t seems sur-prising that more oases of pseudotuberculosis have not been diagnosed and reported. Other diseases such as canary pox have been introduced to British Columbia by imported birds (Stovell and Simon i960). One case of pseudotuberculosis occurring in finches i s considered in this section (Bryner 1906). As noted in Table I there is l i t t l e information given on the aviary history, or on factors suggesting the mode of transmission in many of the literature reports. It seems that extremely high mortality rates were common, although not invariably encountered. Transmission appears to have occurred from bird to bird, either directly or through contaminated food, water, or appliances. The Initiation of an epizootic can evidently be due to transfers of infected birds, contact with rodents, or inoculation of accidentally infected material. During the period discussed, spontaneous outbreaks were also recognized in chickens, FEAR AUTHOR HISTORY OP EPIZOOTIC OR DISEASE INCIDENCE EPIZOOTIOLOGICAI TRANSMISSION FACTORS ASSOCIATED L889 Rieck (l) Dresden May 1888. 1 decomposed bird. Then 3 females and young bird. Last of 200. No observations• -1889 Rieck (2) 60 canaries of unstated number died in 14- days No observations 1903 Wasielewski & Hoffman 1 bird of 3 inoculated with tissue from yellow bunting in malaria studies. Died day .11 PI. a) import with exp, yellow buntings from Holland for ;• Berlin Resulting colony disease worse in Apr & May. malaria studies, b) rats? Former colony thus destroyed 1905 Pfaff Prague 4 birds examined which came from a dealer Cross contact infection at dealer's premises? 1906, Kinyoun. Wash, DC Several birds from dealer were affecial Contact at dealer's premises? 1906 Bryner Germany or Switzerland 60 tiger or butterfly finches and Japanese titmice. 1 cage spread to 3. 22 dead in 1 mo. Lateral spread from cage to cage 1908 Meissner & Schern ex Magdeburg 'Typical'disease (losses not stated) affecting 800 birds Shipped in from Magdeburg 1 9 0 8 Z w i c k - a ) Sept 1904. 27/28 females died. 8 day period. Apparently brought in by Stuttgart b) Males 14- days later. 4-1/4-2 in 14- - 16 days, caretaker who lost own birds e) 6 females, 4 males purchased- in Dec & placed previously.;' 500 - 600 birds in clean cages in adjoining rooms said to have died around d) In 3-4 days surviving female (a) placed with Stuttgart during this period new females. All died but new males. __. - 1914 Ziiss July 1912, 44 birds of unstated number died in G-iessen three weeks ; ] - 1959 Stovell 5 month period Feb to June 58, 3 major separate 2 cases rodent contact known BC epizootics in aviaries (reported herein) 90 - • Dead rodents in flight cage 100 fo mortalities in one case evidence of •severe field mouse population • drop at this time  TABLE I HISTORY AND EPIZ00TI0L0GY OP PASTEURELLA PSEUDOTUBERCULOSIS OUTBREAKS IN CANARIES AS REPORTED BY VARIOUS AUTHORS 00 9 and toward the end of the per iod , i n turkeys. Poss ib ly the incidence i n domesticated b i rds during that per iod suggests e i ther a greater degree of exposure or maintenance of a lower standard of san i ta t ion than i s now the average i n poul t ry and •_aviary husbandry. C l i n i c a l l y , there appear to have been few d i s t i n -guishing features of pseudotuberculosis reported i n canaries . This i s probably due i n part to the small s ize of the b i rds and the d i f f i c u l t y i n handling them, (Table I I ) . The post-mortem les ions recorded are consistent though fragmentary. I t appears that i n bi rds the presence of t y p i c a l 'm i l l e t - s i z ed ' nodu l e s ' i n l i v e r and spleen i s patho-gnomonic. Where the l a s t few surv iv ing birds of an outbreak are the only specimens examined, the diagnosis may be more d i f f i c u l t . For purposes of comparison, some of the f indings from epizoot ics i n canaries i n B r i t i s h Columbia are recorded i n Table I I I . No descript ions of the histopathology of t h i s condi t ion i n canaries were found i n the l i t e r a t u r e . Pal laske (1933) has described the les ions noted i n one canary which was Infected paren te ra l ly . . The problem of d i f f e r e n t i a l diagnosis of pseudo-tuberculosis i s not serious i n cage b i rds , i n contrast to the YEAR AUTHOR CLINICAL OBSERVATIONS PATHOLOGY NOTED 11M SPUNTAJMEOUS DISEASE IN CANARIES Lung•- - • L i v e r - Spleen Bowel Other 1889 Rieck Dresden Quiet, Anorexia Diarrhoea Grey-yellow n e c r o t i c spots C a t a r r h a l e n t e r i t i s D i s c o l o r e d skin,neck breastjabd 1 9 0 3 Wasielewski & Hoffman B e r l i n I n f e c t i o n s followed use of contaminated inoculum. No observations. I f disease- three days c l i n i c a l l y : As spleen Swollen but l e s s Many yellow abundant nodules Nodules often bump up from s u r f a c e * Died 11 days P I . Yellow spots on & i n breast muscle 1 9 0 3 P f a f f Prague Anorexia, diarrhoea sleepiness ^ b i r d s y e l l o w i s h - -white spots E n t e r i t i s 1906 Bryner S w i t z e r l a n d No observations Pinches Some had . l e s i o n s P o s i t i v e f o r l e s i o n s Some had pancreatic l e s i o n s 1906 Kinyoun Wash, DC No observations Did not see b i r d s a l i v e In some Catarr h a l exudate upper a i r passages Y e l l o w i s h nodules p r o j e c t i n g from-s u r f a c e * Some had e n t e r i t i s 1908 Meissner & Schern ex Magdeburg No observations Raised nodules* Some had Di p h t h e r i c membrane 1908 Zwick S t u t t g a r t No observations Grey-yellow r a i s e d noiiles 1914 Zeiss Giessen No observations Hypertrophy Haem E n t e r i t i s Cardiac Haem * Findings i n b i r d s examined i n Vancouver Area TABLE I I CLINICAL OBSERVATIONS AND PATHOLOGICAL LESIONS NOTED BY VARIOUS AUTHORS IN CANARIES SPONTANEOUSLY INFECTED WITH PASTEURELLA PSEUDOTUBERCULOSIS o YEAR AUTHOR CLINICAL OBSERVATIONS PATHOLOGY NOTED IN SPONTANEOUS DISEASE IN CANARIES Lung Liver Spleen Bowel Other 1958 The _author Depressed Very Quite Almost all Enteritis Breast Vancouver Ruffled occasional frequent cases in young muscle 2 out of 3 No singing caseous yellow numerous birds very outbreaks Hop around at times areas or raised yellowish Some had occas occurring Usually continue nodules millet- . nodules caecal thymus w i t h i n eating sized often nodules area reach of Usually utter hoarse nodules giving or Caseous l a b o r a t o r y chirps No extreme abscesses material Diarrhoea in young hypertro phyfhypertrophy occasion-birds usually ally TABLE I I I CLINICAL OBSERVATIONS AND PATHOLOGICAL LESIONS NOTED BY THE AUTHOR IN CANARIES PROM SPONTANEOUS EPIZOOTICS OE PASTEURELLA PSEUDOTUBERCULOSIS INFECTION IN BRITISH COLUMBIA 1 2 case with the disease in rodents. Generally, the c l i n i c a l picture i s of l i t t l e help; some birds are depressed and do not eat, others may be active and w i l l continue to eat or attempt to eat until shortly before death. Many, but not a l l birds develop diarrhoea late in the course of the disease. On autopsy i t i s necessary but not d i f f i c u l t to differentiate pseudotubercu-losis from other diseases characterised by caseating granulo-matous or necrotizing focal lesions; such as l i s t e r i o s i s , enterohepatltis,vlbrionio hepatitis, and others. In addition, care has to be taken to differentiate various types of oatarrhal enteritis such as salmonellosis and coccidiosis. These minor d i f f i c u l t i e s were evidently encountered by early reporters of pseudotuberculosis in canaries; however, apart from some d i f f i c u l t y in naming the condition, the t r a i l was by no means lost at any stage. Malassez and Vignal (LSS4-) named the condition "zoogleic tuberculosis" in the f i r s t dis-covery in the guinea pig. Several reporters apparently gave no name to the condition (Zurn (Rieck 1SS9) (Wasielewski and Hoffman 1903) (Pfaff 1905) (Zwick 190g). In 1S91*- Preisz declared the oausatlve organlzm in birds to be the same as the pseudo- tuberculosis of guinea pigs and rodents. "Infectious necrosis' was the term used by Meissner and Schern in 1903. In 1914- Ziess classed his causative organism along with those of a number of earlier authors, in the 'haemorrhagic septicaemia group', and Beck and Huck (1925) referred to the disease in canaries and 13 turkeys as 'paracholera'. Lerche, who worked with both avian and mammalian strains, and Truohe and Bauche (1929) who worked with turkeys, were a l l of the opinion that the disease should be named 'para-pest' because of i t s close similarity to Pasteurella pestis. Because of possible confusion with the disease 'fowl pest', Lerche did not actually use the name 'parapest', but rather called his disease 'paracholera 1 after Beck and Huck. In succeeding years the disease gradually became referred to as •pseudotuberculosis'. Budgerigars (Shell Parakeet) No reports Involving budgerigars were found in the literature. A personal communication with a pathologist who was at that time in a hospital in London, England (Colbeck 13ko) disclosed that a significant number of deaths oocurred in a budgerigar aviary owned by one of the hospital staff members. The lesions in the birds were classical for pseudotuberculosis and the causative organism was isolated and identified. No v i s i t was made to the aviary, so we have no observations on 1 epizootiology or transmission. Further observations on the 1 The owner stated that she suspected the deaths of the birds to be partly a result of their "being frightened" by owls which swooped over the fl i g h t cages frequently. Pseudotuberculosis in owls in Sweden has been reported by Thai ( l 9 6 l ) . Im-possible susceptibility of budgerigars w i l l be found in a later section dealing with pathogenicity for experimental animals. Finches and Other Captive Hard-Billed Birds One outbreak of pseudotuberculosis in finches has already been covered in part by Bryner (1906). No other re-ferences to the disease in finches have been found in the l i t e r a -ture for the next half-century. In 195^, Thai l i s t e d finches as the source of two of his avian strains. Later, in 1959, Keymer stated that pseudotuberculosis is probably the commonest bacterial agent to cause disease among captive hardbilled birds (especially canaries) in Great Britain. He goes on to state that he has also diagnosed the condition in a Cuban finch (Tiaris canora) and in a wild snow bunting (Pleotrophenax n i v a l i s ) . Langford in i960 diagnosed the condition in a single bull finch in British Columbia. According to the owner, the bird died suddenly with no previous signs of il l n e s s . As can be noted In Figure 7 (Key) finches were present in one oanary epizootic in B.C. However they remained unaffected. 15 F r e e - l i v i n g or Free-ranging Birds In general , t h e l i n f e c t i o n i s thought to be common i n w i l d birds at l eas t i n some areas such as the B r i t i s h I s l e s (Keymer 1959) . Some authors (Hutyra, Marek, and Manninger 194-6)' consider that the in fec t ion i s present i n many countr ies , and i t s occurrence i s so common i n nature that i t s in t roduct ion i s not necessary for the disease to occur. These authors incriminate infected faeces of b i r d s , i n causing spread of the disease. Pigeons and. Doves As can be noted i n Table IV , the Infect ion has been reported frequently i n both sporadic and epizoot ic form. Quite poss ib ly pigeons andc. doves may be the most important avian species Involved i n na tura l epizoot ics leading to subsequent in fec t ion i n domesticated species and man. Of p a r t i c u l a r in teres t would be any findings per ta in ing to the r e l a t i o n s h i p , i n various areas, between.wild b i rds and w i l d rodent ep i zoo t i c s . In the area of England covered by Clapham (1953) detectable i n f ec t i on was apparently confined to b i rds during the study per iod . Recently Paterson and Cook (1962) reported on 'repeated re - in t roduc t ion of pseudotuberculosis to a large guinea p i g colony by use of greenfeed contaminated with the excreta of infected wood pigeons, (Columba palambus)' . TEAR AUTHOR CIRCUMSTANCES PATHOLOGY AUTHOR'S CONCULSIONS 1916 Dolfen Hannover Dead 3 yr old carrier pigeon Typical nodules in spleen liver lungs Similar organism isolated by Pfaff (1905) & Meissner &' Schern (1908) from canaries 1916 Meder* Germany 7 pigeon deaths in recent flock add'ns as above as above 1 9 2 8 B e c k Leipzig 1 pigeon Miliary necrotic lesions in spleen and liver Identical to 1 canary and 5 turkey strains isolated at Leipzig 1923-24 1933 Pallaske Leipzig Not stated L934 Les Bouyrie Prance Worked with 2 strains from spontaneous infections s 20 sick. Flock of 30. 10 deaths.. Pronounced lameness, anorexia.Die 3-8 days after clinic-al onset 2 birds autopsied 1 hepatic & splenic nodules; 1 nodules in duodenum 1951 VanDorssen 2 diseased pigeons organism isolated Suggested organism closely related to Shigella pfaffi 1953 Clapham 3 month epizootic in Hampshire stock doves. 1 wood England pigeon of 17 examined found infected Small caseating nodules in liver & spleen. Both organs slightly enlarged Did not seem to be a rodent epizootic in the area at that time 1950 to 1954 19oT Marthedal & Villing Denmark 7 outbreaks recorded in pigeons Original paper not obtained Patterson & Cook Southern England Flocks of wood pigeons Columba palambus found grossly affected and excreting numerous P. pseudo organisms Not stated whether deaths recorded Repeatedly infected guinea pig colonies by faecal contamina-tion of green feed * Cited by Dolfen TABLE IV PARTIAL FINDINGS OP VARIOUS AUTHORS RELATING TO SPONTANEOUS PASTEURELLA PSEUDOTUBERCULOSIS INFECTION IN PIGEONS & DOVES H ON 17 Pheasants and Other Galliformes There are several reports of infection in or work carried out on strains from species of this group, particularly in the pheasant (Hutyra et al.) (Truche and Bauohel933> Tot-*ire Hippolitl 194-2; Karlson 194-5; Clapham 1953; Thai 195*0. Hutyra et a l . have classed the disease reported by Klein (1921) as pseudotuberculosis. Truche and Bauche, in France, noted that three or four deaths occurred in a pheasant flock over an eight-day period. Small grey spots were noted on the liver surface and the bacterium was isolated from l i v e r , blood, and bone marrow. Both Karlson and Thai are cited here because they each worked on partridge strains. Clapham Isolated the organism from a pheasant, a partridge, and a Bobwhite quail. Blackbirds and Crackles A complete description of a case of pseudotuberculosis in a blackbird has been contributed by Beaudetta (1940) . This report was accompanied by an excellent review of the infection in birds. Beaudette noted that apart from the canary episode re-ported by Kinyoun (1906) (Table I) (unconfirmed baoteriologically, but doubtless pseudotuberculosis — PLS), his case in the black-bird was the f i r s t reported in any bird in the United States. The bird was thin, lame, and suffering from diarrhoea. The l i v e r was found to contain one yellow nodule and a number of grey ones. 18 Clark and Locke (1962) reported on two outbreaks of pseudotuberculosis occurring in purple grackles (Qulscalus  qulscula) in Maryland in 1959 and i 9 6 0 . The second event was a major epizootic occurring during early spring in an ioterid roost which held up to one million birds during the winter months. Isolations of Pasteurella pseudotuberculosis were made from dead and sick birds (shot). Gross and histopathological lesions were well desoribed. The l i v e r , spleen, lungs, breast and other skeletal muscles were involved with white or yellow pseudotubercles. The mortality was calculated to he high, and grackle carcasses were found in large numbers along roads and around buildings up to mile away from the roost. Various factors of late, winter stress were thought to contribute to such epizootics. Other Free-living Species of Birds Although the information is by no means complete, Table V has been prepared by l i s t i n g some of the reports which can be found in the literature, involving among other species buntings, larks, waxwings, Jackdaws, rooks and swans. Apparently a wide spectrum of avian hosts can be affected by the nodular lesions of P^ pseudotuberculosis, and one i s tempted to suspect that many cases of this disease must occur which are not investigated, identified or reported. YEAR AUTHOR LOCATION SPECIES AFFECTED AND DETAILS PATHOLOGY 195^ Marthedal & Villing Denmark Snow Bunting 19 59 Keymer England Snow Bunting 1903 Wasielewski & Hoffman Germany Yellow Buntings from Holland Not noticed apparently 1953 Clapham Hampshire England One lark found dead Liver showed signs close network of caseating fibers. Spleen showed two bulging nodules of pinhead size 1956 Marthedal & Vi ailing Denmark Waxwings 1.953 Clapham Hampshire England Jackdaw and Rook 1935 Truche Normandy France Swan — 2 died Enteritis; nodules in liver and spleen Cardiac haemorrhage Renal congestion 1937 Urbain & Nouvel France Toucans — 2 varieties In captivity for some years 1954 - Sweden Paradise Birds Strains only ' " ' mentioned TABLE V PSEUDOTUBERCULOSIS IN VARIOUS WILD BIRDS PARTIAL DATA OF SEVERAL AUTHORS H 20 Domesticated Fowl Used for Human Food As w i l l be noted in Table VI, a number of outbreaks of pseudotuberculosis have occurred in chickens in various European countries, and one incident i s recorded here involving a flock of chickens i n Alberta. Severe outbreaks of pseudotuberculosis have occurred in turkeys in Oregon, and the condition has also been reported in California (Table VII). The condition was common in Europe in the early part of the present century, and i t may s t i l l occur there, but perhaps is reported less. The findings of the various authors suggest that under certain adverse conditions, turkeys must be highly susceptible to pseudotuberculosis. Perhaps the current sanitary level in turkey management 1B less l i k e l y to predispose to epizootics even i f the organism should be there, due, for example, to the presence of commensal or wild rodents. There have been at least two reported oases of pseudotuberculosis in ducklings. Boquet (1937) attributed one of these to Truche and Bauche (1930). Another case was reported by PlasaJ (1929). It seems that the disease is probably not an economic problem in this species. YEAR AUTHOR CIRCUMSTANCES & INVESTIGATION PATHOLOGY AUTHOR'S COMMENTS 1885 No card France Same organism as Malassez & Vignal's Bacillus 1897 Woronoff & Sineff . Germany 1926 Tottire Hippolito Italy 31933 Truche & Bauche ; . Praxrce Plock of 50* Several vaguely sick. 8 dead one morning and two the next.~ Exudate in nares Non-pathogenic for Yellow gelatinous fowls IV. Considered material in pericardial P.pseudo required a sac. Peritrachealoedemapredisposing Nodules in kidneys infection Slight enteritis 1937 Truche & Isnar.de Slow moving infection flock of 57• 11 deaths in 11 months. * Nodules in intestine P. pseudo isolated & mesentery noted by from bone marrow Aisne region Prance 12 pullets died at age 3 mos. owner. He submitted feet to lab. Custom-ary in Prance 1939 Schafer Konisberg Germany INfections in young chickens 19^7 Karlson Sweden Worked on one chicken, strain of a total of 13 avian source isolates 19^8 Mira Spain 1925 Denmark Importation of live poultry species banned due partly to pasteurella infections continued overleaf T A B L E VI PARTIAL LIST OF AVAILABLE REFERENCES IN THE LITERATURE CONCERNING PSEUDOTUBERCULOSIS IN CHICKENS H YEAR AUTHOR CIRCUMSTANCES & INVESTIGATION PATHOLOGY AUTHOR'S COMMENTS 1954 Marth.ed.al Denmark 'Sporadic chronic or subacute cases s t i l l noted i n fowl'. "4~0 chickens died i n fl o c k of 4-5 month old birds Some cases of Pasteurella were Pseudotuberculosis I 9 6 0 S t o v e l l & Avery One b i r d examined. M i l i a r y lesions i n l i v e r and hypertrophy of spleen Typical of P.pseudo bacto and by animal "inoculation (canaries and guinea.pigs) i n i t i a l l y fermented sucrose PARTIAL LIST OF AVAILABLE REFERENCES IN THE LITERATURE CONCERNING PSEUDOTUBERCULOSIS IN CHICKENS YEAR AUTHOR . CIRCUMSTANCES & INVESTIGATION PATHOLOGY AUTHOR'S COMMENTS Krage & Weisgerber Germany Outbreak'In 14 turkeys.Chick- Catarrhal enteritis, ens, pigeons and geese in Enlarged spleen, contact were not affected Haem on peritoneum. No tubercles seen Isolated from two turkeys with no gross lesions 1925 Beck & Huck Leipzig Five- strains isolated in 1923 Reported on bacto similar to canary strain in all respects 1926 1927 Lerche Leipzig Outbreak in turkeys. Worked on total of 35 strahs Called Paracholera after Beck & Huck Perhaps better called Parapest 1928 Haupt Worked on rodei;, canary and Germany turkey strains Found no difference in strains 1929 Truche & First case in turkeys in Bauche France. Several flocks. France " Av Mort 14- - 16$ Catarrhal enteritis of duodenum. Degen. of liver & kidneys Miliary foci of nec-. rosis'in liver often, spleen occas., lung Organism could be called B_j_ parapestis and disease called Parapest 1942 Stephan Worked on one turkey strain 1927 Karlson Sweden Worked on ten turkey of 30 avian isolates strains Felt that disease came directly or in-directly from rodents and required pre-disposing factors 1944 Rosenwald & Dickinson Oregon, USA Ten flocks of turkeys infected in Oregon, in 20 month period Fall 1940. 13250 birds involved Also 500 deaths of 2700 poults 10 flocks examined. 122 deaths in mkt wt birds. Most caseation nodules in liver, at times in spleen. Pane and int submucosa showed'greyish i n f i l -tration' 15 isolates made. Most cases occurred in rainy season & birds drank puddles. Liver best organ for isolation but spleen and heart blood too TABLE V I I PARTIAL LIST OF AVAILABLE REFERENCES IN THE LITERATURE CONCERNING PSEUDOTUBERCULOSIS IN TURKElS continued overleaf YEAR AUTHOR CIRCUMSTANCES & INVESTIGATION PATHOLOGY AUTHOR'S COMMENTS 1954 Mathey & Siddle "Calif USA Sporadic case in eight-month old torn turkey 1954" ThaT Sweden Abdominal ascites shrunken cirrhotic liver Pale heart muscle Of 186 strains studied 19 of 32 avian strains were of turkey origin Culture from heart and liver TABLE V I I page - 2 PARTIAL LIST OF AVAILABLE REFERENCES IN THE LITERATURE CONCERNING PSEUDOTUBERCULOSIS IN TURKEYS 25 DESCRIPTIONS OF THREE EPIZOOTICS OF PSEUDOTUBERCULOSIS IN BRITISH COLUMBIA CANARIES Three canary epizootics of pseudotuberculosis occurred in 1953 in B.C. These are described and discussed in terms of the recording of natural conditions and events, the epizootilogical deductions that were drawn, and the laboratory supportive work as far as i t concerned diagnosis. History and Recorded Observations AVIARY No. 1 Date of Epizootic Late December to early April (balance of stock in aviary destroyed April 3, 1958). Location of Aviary The building faced south in a well-drained, sheltered lot in a spacious residential area in south central Vancouver. There were many trees and garden lots close by, Including one immediately adjacent to the back of the aviary which was used for growing vegetables. This lot was f e r t i l i s e d once yearly in the f a l l with cow manure from a dairy farm in Richmond. 26 Design and Condition of Building The aviary was a single-storey, permanent, double (two-flight) aviary constructed of timber and wire and completely roofed with cedar shingles. There was fine gravel on the concrete floor in the flights, a central entrance and servioe passageway gave access. The flights were open to the front and part of the sides (Figs. 1 and 2) . Operational Details and Management Feeding of a commercial canary seed mixture was carried out with the use of two wooden floor troughs which were replenished daily. In mid-winter the caretaker mixed cod li v e r o i l emulsion with dampened seed to a porridge consistency and offered this In f l a t dishes on the broad ledges of the f l i g h t s . For most of the year, chickweed from the vegetable plot was fed onoe or twice weekly to the birds, being pushed into the flight cage wire so that the birds could p u l l i t . Water was supplied in a galvanised trough and replen-ished daily. During the breeding season, extra water dishes were frequently placed on the flight ledges. Because of the f u l l cover and sheltered area, no extra weather protection (suoh as glass screens) was used in the winter. When health conditions were normal, the nest boxes were placed in the f l i g h t . These were attached to the beams and Figure 1 Aviary,No 1 Southerly aspect with entrance to service passageway. Views on this and the following pagees show the aviary involved i n the f i r s t epizootic of pseudotuberculosis in canaries. This building combined f l i g h t s for both canaries and budgerigars. Figure 1 28 posts close to the ceiling of the f l i g h t , thus being sheltered by the overhang. Rodent control was carried out by occasionally setting spring traps and by covering with t i n any holes which were gnawed through the back of the f l i g h t . It was stated by the owner that there had been no rodent problem until the f a l l of the year when the outbreak commenced in December. Stock in Aviary at Commencement of Losses The west fl i g h t contained approximately f i f t y budger-igars, which were managed in much the same way as the canaries, and which suffered no losses or noticeable disease,. (Figure 3). The east f l i g h t contained twenty-nine canaries in late December, 1957- These were mostly hatched in 1956 and 1957. Approximately twenty birds of the older stock were given away to the Stanley Park aviary in early f a l l . Record of Mortalities As w i l l be noted in Table VIII, in a total of twenty-nine birds, ten deaths occurred over an approximately three-month period. The mortality rate climbed to the level of about one death per four days by mid-March. The remaining nineteen birds were destroyed with C0 2 at the beginning of April, and sixteen of these were found to have advanced lesions of pseudotuberculosis mainly involving the spleen (Fig. 4-). Figure 2 Figure 3 Aviary No 1. Flight used for canaries. Note the gravel floor and spacious quarters for approx-imately thirty birds. The elevated feeder at centre was Installed later on the suggest ion of the author. Aviary No 1. West flight housing budgerigars which remained healthy. Note nest boxes, hanging roosts, and placement of feed and water dishes similar to those used in the canary fl i g h t . A v i a r y N o 1. S o m e i n t e r n a l o r g a n s o f i n f e c t e d c a n a r i e s . T h e s e b i r d s w e r e s a c r i f i c e d s i x t e e n d a y s a f t e r m o r t a l i t -i e s b e c a m e h e a v y . O n e n o r m a l a n d i fchree a f f e c t e d s p l e e n s ( m i d d l e r o w ) c a n b e c o m p a r e d f o r s i z e w i t h h e a r t s ( t o p r o w ) a n d g o n a d s ( b o t t o m r o w ) f o r t w o m a l e a n d t w o f e m a l e b i r d s . N o t e t h e p r e s e n c e o f c h a r a c t e r i s t i c y e l l o w n o d u l e s ton t h e s p l e e n s . T h i s l e s i o n , w h i c h i s v i r t u a l l y p a t h o g n o m o n i c ^ , w a s n o t e d p r e d o m i n a n t l y i n t h e s p l e e n s i n t h i s o u t b r e a k . F o u r t e e n o f s i x t e e n b i r d s s a c r i f i c e d s h o w e d t h i s i n v o l v e m e n t . F i g u r e k-Figure 4-BIRD NO. SEX DATE 0E DATE DEATH KILLED C02 CLINICAL APPEARANCE TO CARETAKER PRESENCE OE SPLEEN LIVER LESIONS OTHERS P.PSEUDO ISOLATED Not designated,"but four birds died from Xmas to early March individual b'irds that died usually sick 1-2 days. Gradually whole group depressed. Singing stopped. Not examined 1 E 14 March Depressed, rough 1-2 days pos pos caecal buds yellow nodules isolated 2,3,4 2M IE 16 17 March Depressed, rough 1-2 days 3 pos IE pos 3 neg not attempted 5 29 March Depressed, rough 1-2 days pos pos neg not attempted 6,7,8,9 2E 2M : ' 1 April Slightly dull 3 pos IM neg 4 neg 4 neg not attempted 10-25 8M •8E 1 died 3 April en route to lab Slightly dull 14 pos 2M NEG 3 pos 2M IE 16 neg not attmpeted TABLE V I I I RECORD OE NATURAL LOSSES, BIRDS DESTROYED EOR AUTOPSY, AND POSTMORTEM RESULTS EOR THE EPIZOOTIC OE PASTEURELLA PSEUDO TUBERCULOSIS INFECTION IN AVIARY NUMBER 1 ?:, 32 Discussion of Epizooti©logical Aspects (a) Sanitation was excellent; more birds could have been housed in this f l i g h t without overcrowding. (b) The feeding regimen was adequate and unvarying, although one remote and one less remote possibility of infection through this source should be considered. These possibilities were: contamination of canary seed with rodent droppings before i t l e f t the mill; and secondly, the contamination of chickweed from wild rodents or birds, or from the cow manure used for f e r t i l i s i n g the plot. (c) There had been no additions to the flock for the last eighteen months, except for natural i n c r e a s e s T h e previous f a l l , a group of about twenty adult birds was transferred to a local zoo aviary. No losses occurred in the transferred birds during a three-month Isolation period at the zoo. No subsequent trouble developed in these or contact birds at the zoo during 1952. (d) During the f a l l , many f i e l d mice were seen around the garden in the vic i n i t y of the aviary. Several penetrations were made Into the flight by mice. About two weeks before the f i r s t canary death occurred, two f i e l d mice were found dead one morning in the canary f l i g h t cage. Following the laboratory diagnosis, the caretaker was asked to live-trap or poison rodents for examination. Shortly thereafter he reported that there were 3 3 no longer any signs of mice in the area. Although no mice were examined, the above factors raised the suspicion of the canary disease being a reflection of an epizootic in mice. Contamination of canary seed could have occurred leading to oral transmission of the organism. AVIARY No. 2 Date of Epizootio Late April to July 1 9 5 3 , a period of approximately ten weeks. Location of Aviary The aviary was situated in the sheltered garden of a spacious rural residential lot at Qualicum Beach on the east coast of Vancouver Island. This was a luxury summer cottage with year-round caretaker residence. Design and Condition of Building This aviary (Fig. 5 ) was similar in style and con-struction materials to tbe building involved in outbreak No. 1. The canaries were housed in a flight which formed a horseshoe shape about the service alleyway. The floor had been concreted several years earlier to control rodent entry. Number 1 and Number 2 aviaries belonged to the same owner but were situated about seventy miles apart on opposite sides of the Strait of Georgia. Figure 5 Aviary No. 2. General aspect of building. The stock was comprised entirely of canaries. Figure 6 Aviary No. 2 Close up view of flight cage. This shows feeding trough(above) and water dishes on the floor. As illustrated, many birds prefer to feed from the floor on seeds scattered by the birds above. This seems to be representative of passerine behaviour under natural conditions where a few individuals attack the heads of grasses and grains, while others pick up the fallen seeds. Thus exposure to fecal material by ingestion is increased. 35 Operational Details and Management These were essentially the same for the two aviaries; in fact the caretaker at the No. 1 premises had earlier been in charge at the No. 2 premises. He had built both aviaries and later re-floored them with concrete. Stook in Aviary at Commencement of Losses There was a total of sixty-eight canaries Involved. These were comprised of forty-eight adult birds and twenty young birds hatched during the period of the epizootic. About the middle of May, twelve of the more mature young birds were selected and transferred to aviary No. 1. In the meantime, following the destruction of the birds in the No. 1 Aviary on April 3, the premises had been thoroughly cleaned and disinfected. This, with the addition of rodent control which was also employed, would seem to eliminate the possibility that these transferred birds became infected from the Vancouver environment. There were no budgerigars or other captive birds on the premises of the No. 2 aviary. However, there were crows in the area which came around the fl i g h t cage. During the previous year California quail had been numerous in the area also. 36 Record of Mortalities No exact records were kept by the caretaker. However, since the disease, with or without complications of secondary infections, caused the death of most of the birds, i t was not hard to calculate the percentage mortality. By July 10, deaths .had ceased and there were two birds (3 .6$) l e f t of the f i f t y - s i x adult and young which had become sick. Thus the mortality was 96 .4$ . At one point the caretaker moved many of the sick birds to some old pheasant pens at the opposite end of the garden, thinking that he might modify the ravages of the infection in the remaining birds. Deaths of birds continued unabated in both locations. Of the twelve young birds which were moved to the Vancouver (No. 1) aviary, nine (73%) survived. Shortly after their arrival, the caretaker noticed that two birds were depressed and a third became unusually tame. One of the former and the latter died after about seventeen days, and these were placed in the freezer. Finally, in August the author was again contacted. The two frozen birds were autopsied and the third previously siok bird which was s t i l l not vigorous was sacrificed for autopsy. The two dead birds showed typical lesions of pseudotuberculosis and a pure culture of psuedotuberculosis was isolated. The third showed no gross pathological lesions, and a culture of pooled tissues was negative. The nine birds remaining gave no 37 further trouble, and from these birds the aviary population was built up to twenty birds in about two seasons. Now, five years after the clean-up, there has been no visible recurrence of disease. Discussion of Epizootiologlcal Aspects (a) Sanitation was good and there was no overcrowding in this aviary. (b) There is no reason to suspect the feed'of being contaminated prior to dispensing. There i s a possible exception to this again in the green feed collected from the vegetable garden, which might have been contaminated by the faeces of wild rodents or birds. (c) There had been no additons to the birds other than natural Increase for five years. (d) The area surrounding the garden was the haunt of various species of rodents, galliform birds, and others suscep-tible to pseudotuberculosis. There were crows around, which would settle on the cage and thus doubtless at times contaminate the wire fence with faeces. F i e l d mice were seen at times in the aviary surrounds. (e) There is the remote possibility that the owner or one of his family might have mechanically transferred the infection 38 from the Vancouver aviary, however, i t was not the habit of these people to actually enter the aviary at either location. AVIARY No. 3 Date of Epizootio Mid-June to late August, a period of about eleven weeks. Location of Aviary The aviary was located on a sheltered south slope in North Burnaby municipality of the greater Vancouver area, situated about twelve miles from the No. 1 aviary. The aviary building was surrounded by brush and brambles. Design and Condition of Building The,building was a lightly constructed converted chicken house which had been previously used for housing dogs ( a l l of which were reported to have died). It was a single-storey unlined wooden house of shiplap construction with a tar paper exterior. Half of the south wall of the building could be opened down outside and the spaoe was covered with wire in some places, in other places with glass. Except for an entrance doorway along the south side, the east end of the building was taken up with three semi-outside fli g h t cages. There were many 39 cages spaced out i n bays i n s i d e the b u i l d i n g , two l a r g e Inside f l i g h t s , and a number of small cages hung i n the p o o r l y v e n t i l a t e d space above the window on the south s i d e . A few cages, c o n t a i n i n g ten to twelve b i r d s , were hung on the back of the b u i l d i n g ( F i g . 7 ) . Operational D e t a i l s and Management The a v i a r y was u n s a n i t a r y , overcrowded and i n most p a r t s , p o o r l y v e n t i l a t e d . Rodent c o n t r o l was quite inadequate, and w i l d or commensal rodents were encouraged by a l a r g e p i l e of feed sweepings on the ground o u t s i d e , c l o s e t o the entranoe doorway. Watering dishes were removed from cages, emptied, r i n s e d together i n a p a i l , f i l l e d and d i s t r i b u t e d a t random. Feed dishes were removed, emptied, r e f i l l e d w i t h new feed, and r e p l a c e d a t random. Breeding was c a r r i e d out i n a row of cages next to the f i r s t i n s i d e f l i g h t a t the east end of the b u i l d i n g . For the general layout of cages and groups of b i r d s , see F i g . 1 . Stock i n A v i a r y at Commencement of Losses There was a t o t a l stock of 3°° b i r d s a t the middle of June. These were made up o f s e v e n t y - f i v e n a t i v e Lennets, Weavers, G o c k a t i e l s , Budgerigars, and Finches ( s p e c i e s not reoorded). The balance of 225 b i r d s were e i t h e r c a n a r i e s , mules , 1 Canary cross w i t h (a) Red Sisken, or (b) the F l or F2 (backcross) 4-0 Key to Figure 7 Reconstructed Line Drawing of Aviary Involved i n Ep izoo t i c Number 3 1. F l i g h t "MF" containing t h i r t y mules and factors when the ep izoot ic began. The i n i t i a l losses s tar ted In t h i s f l i g h t about ten days before extension of the disease to other locat ions as marked* . 2. Metal s t r i p along the Inside of the baseboard along the north w a l l which covered rodent holes . The termination of t h i s s t r i p at a point opposite the "MF" f l i g h t had the effect of funneling the rodent entr ies through the back of the b u i l d i n g along a l i n e marked wi th V s . 3. Point of ingress of rodents which then followed a pathway through the MF f l i g h t and under or over the area where breeding pa i r s were kept, to the inside of the av ia ry . 4. Counter area where most of the breeding cages were loca ted . 5. Semi-outside f l i g h t s which contained respec t ive ly : (a) Lennets and two cockat ie l s (b) Weavers and finches (c) Budgerigars, approximately s ixteen i n number. 6. Small cages for i n d i v i d u a l b i rds , suspended i n overheated space above the windows and against the roof on south f ron t . 7. F l i g h t "0" which contained o ld b i rds at the beginning of the ep izoo t i c . These were serviced wi th f i xed seed and water containers, and perhaps for th i s reason remained free of i n fec t ion u n t i l younger exposed b i rds were placed i n the f l i g h t . Four of the bi rds from th i s f l i g h t were t ransferred to the laboratory as part of group I af ter losses commenced In th is f l i g h t . Location of a large dump of seeds and sweepings from the av ia ry . I I P - L C O N ' S T P . U C T t D L-IM6- P P , A W I M C O P AVlAPsY I N V O ^ \ / £ . D [N E - P I X O O J l C N 23 M O P - T H e » u p - M A f e Y , t>-C- JUUk- - A U G U S T 1^ 53 fxj i r -xxxxxxxxxxxxxxxxxxxxxx:> pcx (2) (7) i f — - , - y (1) i I r i t-I _ j „ i i 4 I o (5c) l i <^ nrp-n 4r y P L A M (8) 4 I — 1 i (5c ) i ( 5h)\5'-I SOUTH- m v A T i o ^ APP - i i H / £ 3 F i g u r e 7 . A v i a r y No. 3 housing canaries and other cage b i r d s . 42 1 or red factors . Many of the birds were fledglings of the breeding season in progress. The aviary had been stocked three months prior to the outbreak,from other premises (belonging to the same owner) where no subsequent disease trouble arose. Record of Mortalities Morbidity, according to the owner, was 100$ among the 225 canaries and canary cross birds (mules and red factors). Except for the modifying effects involving the thirty-five birds removed to the laboratory, the mortality was also 100$. The budgerigars, cookatiels, native lennets, weavers and finches were not affected to a l l appearances, and no mortalities occurred. There was an i n i t i a l loss of two or three birds around mid-June. The epizootic came to a peak at the end of June and seventy-five birds were lost between June 26 and 30. By July 15, when news was next reoeived by the writer, the losses totalled 165 (73»3#) o f t h e 225 canary and canary cross birds. At this time twenty liv e birds (designated as Group I, Experiment A) were removed to the laboratory. On a r e v i s i t July 29, ten more deaths had occurred, and fiteen more birds (designated as Group 1 Canary orossed with F3 mule (backcross) 4-3 II, Experiment A) were removed to the laboratory. Fifteen fledg-lings remained at the aviary. These were removed to the outside by the owner in hopes of effecting a modification in the disease course. On September g, the owner informed us that the last of the birds died around the end of August. Thus mortality on the premises was 100 per cent of the 190 susceptible birds which were l e f t there. These figures are summarised in Table IX. Of the thirty-five birds transported to the laboratory, thirty-four died eventually, either of confirmed pseudotuberculosis directly or of i t s apparent after-effeots. Discussion of Epizootiological Aspects Four main factors appear to be significant i f one assumes that rodent contact could have been involved in starting the canary epizootic. There are: the size of the rodent population; the form of dispersal of the birds and rodent contact with them; the poor sanitation practices with regard to food and water; and the general stress on the birds due to breeding activity, poor ventilation, and actual overheating of many parts of the building, particularly during the month of July. An important potential factor was the feeding of paprika to birds in the fl i g h t where the f i r s t death occurred. The f i r s t two of these faotors may be summarised and broken down as follows: (a) Observed presence of large number of mice in and TIME PERIOD NUMBER OP NEW LOSSES CUMULATIVE TOTAL LOSSES AT AVIARY BALANCE IN AVIARY* REMOVED TO LABORATORY** June 12 - 15 (circa) 3 3 222 June 16 - 24 0 3 222 June 26 - 30 75 78 147 July 1 * 15 87 165 60 July 15 165 40 20 (Group I) July 16 - 29 10 175 30 July,29 175 15 15 (Group II) July 30 - August 31 (oiroa) 15 19Q 0 TOTAL 190 190 0/225 35 * Of the 225 canary and canary-cross birds present which appears to have been the only group susceptible to the degree of exposure which occurred ** See Experiment A TABLE IX RECORD OP MORTALITIES OCCURRING IN NO. 3 EPIZOOTIC OP SPONTANEOUS r:rn- PASTEURELLA PSEUDOTUBERCULOSIS INFECTION IN CANARIES 45 around the premises. This would have been predictable in light of: (i) The pile of floor and cage sweepings with discards from feed containers a few feet from the entrance. ( i i ) The presence of uncovered food in bags and cans on the floor. ( i i i ) The ample cover for rodent breeding right against the back wall of the inside of the building. This fact was confirmed when the building was viewed after removal of the cages. (b) Form of dispersal of the birds and rodent contact. It w i l l be noted in the accompanying chart that the unaffeoted species of birds were housed in the semi-outside flights at the east end of the building. There were thirty selected mules and factors which were in the inside flight adjacent to their cage, (Flight MF, Figure 7). These birds were being fed a gastro-intestinal i r r i t a n t , paprika (supposedly to Improve the colour for show purposes). This inside flight by chance straddled the route of ingress of the rodents to the house. A baseboard metal strip along the rest of the back of the building restricted the rodents' entry to this point. The epizootic f i r s t made i t s appearance in these birds. An additional factor not previously mentioned was the presence of about six or eight loose birds flying in the aviary, which could have spread the infection through their droppings. Their presence was noted on the July 15 inspection. b6 Summary of Epizootiologioal Considerations for the Three Affected Aviaries The main epizootlological factors to be considered in light of the histories of these aviaries are f e l t by the author to be: (1) The presenoe of significant numbers of rodents, especially f i e l d mice, and in particular any evidence of sickness, or of their entry into f l i g h t cages where canary deaths subse-quently occurred. (2) The presenoe of other wild species known to be susceptible to psuedotuberculosis which came around or came in contact with the f l i g h t oages. Wild birds are an example. (3) The development of generalised stress in the presenoe of specific stress mechanisms (e.g. feeding of paprika -a bowel irritant) which would act as a predisposing factor to the occurrence of frank disease in the presence of the causative organism. It is apparent that rodents, even dead ones, were in close contact with the canaries in aviary No. 1. Also reasonable is the assumption that contact was equally close in the MF fl i g h t of Aviary No. 3 (where deaths commenced). In the case of Aviary No. 2, the evidence of mice is more circumstantial than actual. The environs evidently harbored a considerable range 4-7 of w i l d l i f e including avian species known to be susceptible to pseudotuberculosis, such as galliformes and crows. It seems reasonable to attribute some importance to the winter period (Aviary No. l) the reproductive period (Aviary No. 2) and the over-heating and poor ventilation (Aviary No. 3)» when considering general factors of stress. One specific stress factor, the feeding of paprika, also occurred in Aviary No. 3« Laboratory Diagnosis of Pseudotuberculosis Bacteriological Diagnosis Introduction Meyer (1959) states that the term 'pseudotuberculosis' must be reserved s t r i c t l y for infections caused by Pasteurella pseudotuberculosis. In spite of a total of at least ten synonyms whioh are s t i l l quoted by various authors (Meyer 1959; Bergey 1957; Stafseth 1959; Zinsser 1957), ^he actual identification of the organism now known universally as P^ pseudotuberculosis is not considered d i f f i c u l t by most bacteriologists. The d i f f i c u l t i e s of differentiation of this organism from Pasteurella pestis and Pasteurella multocida are overcome by adequate biochemical characterisation, inoculation of experi-mental animals Including the albino rat; and demonstration of motile qualities for the pseudotuberculosis organism. This 48 author has experienced few aberrations while identifying avian strains. However, this i s not the case with a l l those of mammalian origin; for example, those from British Columbia 1 chinchillas. In recent years, various leading authorities (Meyer 1962; Murray 1962) have expressed the view that the Easteurellas are inadequately designated, and that some familial re-grouping may be desirable for certain members, including pestls and P. pseudotuberculosis. Bergey (1957) makes a .positive contribution to this problem by l i s t i n g a separate species designated Pasteurella p f a f f l (Hadley 1918) which has been considered for many years to be identical to P^ pseudotuberculosis (Haupt 1934-). In light of recent advances in bacterial genetics, the separate designation for a single strain of an organism studied thirteen years after i t s isolation (Pfaff 1905) appears unjustified. Primary Isolation Routine methods were used throughout with caseous material or ground saline emulsion of tissues being plated onto blood agar and sometimes MacConkey's plates. Typioal 1 This experience has also been encountered in Denmark with some chinchilla strains which are active sucrose fermenters (Knox 1963). / 4-9 P. pseudotuberculosis colonies were picked with the use of a hand lens and placed in beef heart infusion or Robertson's meat mash and incubated at 3 7 ° C for biochemical identification or at 22°C 1 for demonstration of motility. Identification of Pasteurella Pseudotuberculosis (a) Colonial morphology (l ) On nutrient and blood agar. The appearance of the colonies was followed from 24 to 72 hours. During this period, the appearance progressed from low convex to umbonate; from smooth and shiny to granular (sometimes coarsely so); from being completely translucent to grayish yellow and opaque; and from having entire edges to showing effusive edges with radial striations stretching back to the umbonate centre. ( l i ) On MacConkey agar plates. On this medium, the colonies were more inclined to show granularity at twenty-four hours; were more effusive at the edges; and were inclined to lyse after 72 hours. 1 Current practice is to completely avoid incubation at 37 GC and recent isolations have been carried out at room temperature. 50 (b) Staining characteristics (i) Grain's stain. The cocco-bacilli, the short rods, and the few filaments were uniformly Gram-negative> variably bi-polar. ( i i ) Wayson's Plague Stain. The bacillus uniformly showed marked bipolar!ty with characteristic banding in the filamatous forms. Swelling (perhaps due to use of the methyl hydrate fixative) was common with many of the cocco* bacillary forms so that the result of the staining was to give a hamburger-like appearance. ( i i i ) Acid-Fast Staining. A slight degree of aoid fastness could be demonstrated in a l l the oanary isolates with the use of Cook's acid fast method (see Appendix) and this quality was further accentuated with the use of Macchiavello's rickettsia stain. (c) Motility Nine strains and two substrains of P^ pseudotuber- culosis were tested for motility of 25 per cent or better by repeated subculturing in beef heart infusion broth at room temperature (20 to 22°C). These included seven type-strains received from Dr. W. Knapp of Tubingen, Germany; three strains Isolated from two canary epizootics in Brit i s h Columbia (Aviaries No. 2 and No. 3); and one chinchilla isolate from British Columbia. 51 Eight strains were motile after two transfers in beef heart infusion broth. One other required four passages. The two remaining strains were not motile after seven passages; however, i t was noted that they were both inclined to roughness, and since both were substrains, the results were considered satisfactory! (d) Growth characteristics in broth Fresh isolates gave a uniform turbidity for one or two days in beef heart infusion broth, and to a lesser extent in Robertson's meat mash. Following this, the broth usually cleared, leaving a ring at the surface and a heavy flocculent sediment. Isolates from two of the three aviary epizootics yielded clearly motile forms at 22°0 (see above). (e) Biochemical alignment The strains Isolated from canaries aligned well with the reports in the literature for both carbohydrate fer-mentations and for other biochemical tests (see appendix for l i s t of results and accompanying key). (f) Serological typing Isolates from the No. 1 and No. 2 aviaries were submitted to Dr. W. Knapp at Tubingen, Germany, and were re-ported to belong to Type I of ^  pseudotuberculosis. 52 Pathogenicity for Experimental Animals Guinea pigs and canaries were found to he susceptible to Infection with the Isolated strains of P^ pseudotuberculosis, the latter by oral as well as parenteral routes. A budgerigar which was dosed orally was found dead with typical gross lesions twelve days later. White mice were found to be comparatively resistant, and albino rats completely resistant, to the strains isolated. In some cases, as when dosed with ground-up canary spleens, the mice died in twenty-four hours or less, presumably due to the carry-over of endotoxin. One budgerigar dosed paren-terally died in the same way. Six-week-old chickens were found to be almost completely resistant to both oral and parenteral inoculations of strains pathogenio for canaries and guinea pigs. In one case out of three, a guinea pig was success-fu l l y infected with intraperitoneal inoculation of canary faecal material, and was k i l l e d and cultured at fourteen days post-inoculation. The same faecal material f a i l e d to yield the strain by culturlngj however, after this occurrence, significant improve-ments were made in the technique for the culture of canary faecal material. 53 Pathology Diagnosis Gross Lesions A total of twenty-five autopsies were conducted on birds from the No. 1 Aviary epizootic. Six of these were natural deaths, the remaining nineteen were destroyed with COg. Of thirteen females and twelve male birds, three destroyed males only showed absence of gross lesions of pseudotuberculosis. Total organs affected of birds with lesions were: spleen - 22 (100^)J l i v e r - 4 (18%); and caecal abscesses - 1 (4-. 5#). The pathognomonic lesion appeared to be the classical pin-point to millet-sized yellowish tubercles in the spleen. In some cases the l i v e r was similarly affected. The presence of a primary detectable bowel lesion, which appeared in gross, suggested that other mioroscopic bowel lesions may ocour, thus serving as a portal of entry for spread to the spleen. The presence of liver nodules suggested a sequel to a bacteremio phase. For Aviary No. 2, only two birds were examined. These birds showed the presence of typical pseudotuberculosis in both spleen and l i v e r . The gross pathological lesions recorded for birds transferred to the laboratory from Aviary No. 3 are l i s t e d in Tables XI and XII. 5* Several birds with splenic tubercles and one with l i v e r tubercles were examined culturally in making the i n i t i a l diagnosis. For the birds transferred to the laboratory, more observations were recorded with the aim of correlating gross pathological lesions with a b i l i t y to culture pseudotuberculosis, (Table XII). Histopathologlcal Changes The structure of the pseudotubercle was studied with the aid of paraffin sections stained with haematoxylin and eosin. Of those spleens and livers which were examined from f i e l d cases, .nail showed a marked granulomatous response. Mono-nucleic macrophages predominated in the cellular mass. Scattered about were numerous small areas of caseation necrosis, eosino-ph i l i c in tone, and with areas of blue smudging probably due to degenerating masses of bacteria. The oellular response was more diffuse about the necrotic f o c i in the spleen than in the l i v e r . In the latter organ, intense cellular cuffing could be noted, (Figs. 8,9,10,11). Kupjpfer oells were absent, doubtless due to their cytometamorphosis to form larger macrophages. The liv e r cords were completely disrupted with marked vacuolar de-generation of the hepatic oells. The appearance of the macro-phages suggested intense bacteriophagia although clearly defined microorganisms were not v i s i b l e in the way noted in the experi-mental disease (Fig. 56). Extensive haemorrhages were noted in H & E 150X F i g u r e g A v i a r y N o . 1. S p l e e n f r o m s p o n t a n e o u s c a s e o f c a n a r y p s e u d o t u b e r c u l o s i s . T h i s l o w p o w e r p h o t o m i c r o -g r a p h s h o w s n e c r o t i c f o c i s u r r o u n d e d b y a d i f f u s e m a s s o f m a c r o p h a g e s . H & E 500 X F i g u r e 9 A v i a r y N o . l . S p l e e n f r o m t h e c a s e i l l u s t r a t e d i n F i g u r e g. M o r e details o f t h e m a c r o p h a g e s c a n b e s e e n u n d e r h i g h p o w e r . Figure & , Figure 9 H & E 500X Figure 10 Aviary No. 3» Liver from spontaneous case of canary pseudotuberculosis. This photomicrograph shows a discrete pseudotubercle. H & E 500X Figure 11 Aviary No. 3* A*1 aggregation of pseudotubercles in the liver illustrated in Figure 16. I# these photomicrographs i t can be noted that the cellular reaction i s denser than in the spleen (Figures 9 and 10), and more inclined to cuff the necrotic area closely. There is a diffuse degeneration of hepatic ce l l s . F i g u r e 10 F i g u r e 11 57 the spleen along lines of fracture due to structural weakening of the organ. 58 EXPERIMENTAL STUDIES ON THE TRANSMISSION,. FAECAL EXCRETION RATE AND PATHOLOGICAL FINDINGS OF PSEUDOTUBERCULOSIS IN CANARIES When the epizootic commenced in Aviary No. 3, labora-tory studies were carried out on canary pseudotuberculosis, to increase the general knowledge of epizootiology and pathogenesis of the condition. Two groups, totalling 35, o f the affected canaries were transferred to the laboratory and the progress of the disease was studied. This work is reported as Experiment A of this section. As a result of the above study and of a concurrent examination of the literature, a number of question's were raised relating to both epizootiology and pathogenesis. It seemed necessary to create experimental pseudotuberculosis by the (assumed) natural, oral route in laboratory canaries to attempt to answer these questions, along with others which were raised by studying the epizootics in the three affected aviaries. Attempts to arrange the transmission of pseudotuberculosis by contact from experimentally infected to healthy birds were carried out f i r s t (Experiment B). This work was followed by a study of birds infected experimentally per orum (Experiment C). 59 Several of the questions are l i s t e d for which cl a r i f i c a t i o n was sought: (a) Do most canaries become infected from each other during spontaneous epizootics or from a common source (such as rodent excreta)? (b) In the event of recovery, do canaries remain as chronic carriers and excretors of P^ pseudotuber- culosis? tc) What aspeots of transmittance or of pathogenesis account for the extremely high mortality rate which i s apparently common in canaries (close to 1 0 0 $ ) and is almost without precedence among bacterial diseases? (d) Do Infected canaries pass significant numbers of viable P. pseudotuberculosis in their, faeces, eMen thougli the gross pathological lesions are usually limited to spleen, li v e r and occasionally breast muscles? (e) What i s the significance, in terms of faecal excretion of the cecal abscess lesion which was noted on two occasions in spontaneously infected canaries (Nos. 1 & 3 Aviaries)? Materials and Methods General Housing Conditions for the Experimental Periods Depending upon the purpose of the experiment, cages of widely varying design were employed and these are described in detail as each experiment i s discussed. In the case of Experiment A, the primary objective was to give quarters with maximum comfort and space for the birds while the natural disease was being studied in the laboratory. In the case of Experiment B 6o involving contact infection attempts, two widely contrasting levels of sanitation were set up, with birds separated only by a common barrier of wire cloth. For Experiment C, with birds experimentally infected by the oral route, i t was considered essential that the twenty-four birds should be housed close to-gether, so as to be in a uniform environment yet completely in isolation one from the other from the point of view of direct spread of faecal or food materials. The faecal material had to be collected as an entire twenty-four hour sample, complete and without cross-contamination. For these reasons a battery cage was constructed with solid walls. General Bnvironmental Sanitation for Experiments For Experiment A, every precaution was taken to control what was f e l t to be a hazard for human infection. The room air, faecal material and water containers were a l l sampled culturally, q|uarternary disinfectants were used on a l l surfaces and preliminary washing was carried out in the sink in the room with Roccal disinfectant before any equipment was moved out for st e r i l i z a t i o n . For later experiments, carried out for the pur-pose of faecal culturlng, a complete avoidance was made of using disinfectants in the experimental room. A l l s t e r i l i s i n g of materials that could not be discarded was carried out with an instrument s t e r i l i s e r or by placing materials in plastic palls and using chemical st e r i l i z a t i o n in an adjoining room. A l l 6 l spilled seed was brushed up several times daily and the con-crete floor flushed with copious amounts of cold water once daily. Feed, Water, and Supplements For Experiment A, a mixture of three parts commercial plain canary seed and one part mixed millet was used. This was approximately the same formula as had been in use at the source aviary (No. 3). This did not include the addition of paprika. Towards the end of the experiment, the millet supply was dep-leted and this fact probably had bearing on the results of the experiment, and of Experiment B also, where again the plain canary seed was in use. For Experiment C, a more complex mix-ture of seeds (commonly recommended in this area) was in use, while the birds were on hand. This was slightly modified when the faecal culturing was actually in progress. (See Appendix). Feed was offered in open dishes (cans) for Experiment A (Figures -I.2r?13«Ji, and for the low-sanitation section of Experiment B. For the high sanitation (top feeding) section of Experiment B, and for Experiment C, clear polystyrene fountain-type dispen-sers and white plastic hook-on cups were used (Figures . 17-2Q). Tap water from the Vancouver municipal supply was used. For Experiment C, the water dispensers were emptied daily at the time for faecal collection and thoroughly rinsed individually 62 u n d e r a j e t o f c o l d r u n n i n g w a t e r s o t h a t t h e c h a n c e o f b a c -t e r i a l I n f e c t i o n c a r r y o v e r f r o m d a y t o d a y w a s m e c h a n i c a l l y r e d u c e d t o n e g l i g i b l e p r o p o r t i o n s . C u t t l e b o n e w a s s u p p l i e d i n a l l e x p e r i m e n t s . I n E x p e r i m e n t C , a s m a l l p i e c e o f b r o k e n c u t t l e w a s k e p t i n e a c h f e e d d i s h . I f t h e b i r d s t h r e w i t o u t , i t w a s r e p l a c e d f r o m s t o c k d a i l y . N o . 10 g r a d e g r a n i t e g r i t w a s s u p -p l i e d i n o p e n c o n t a i n e r s e x c e p t f o r t h e t o p f e e d i n g s e c t i o n o f E x p e r i m e n t B a n d f o r E x p e r i m e n t C . I n t h e l a t t e r c a s e s , t h e g r a v e l w a s s u p p l i e d i n t h e p o l y s t y r e n e f o u n t a i n - t y p e d i s p e n s e r s . N o o t h e r s u p p l e m e n t s w e r e u s e d . A l t h o u g h t h e a u t h o r c o n s i d e r s t h a t c a g e - b i r d n u t r i t i o n i s p r o b a b l y i n a d e q u a t e u n d e r t h e s e c o n d i t i o n s , i t w a s f e l t t h a t n o a t t e m p t s h o u l d b e m a d e t o f a v o u r t h e e x p e r i m e n t a l g r o u p s w i t h b e t t e r n u t r i t i o n a l c a r e t h a n w a s c o m m o n l y p r a c t i c e d b y c a n a r y a v i a r i s t s i n t h e a r e a . I d e n t i f i c a t i o n o f t h e b i r d s S o m e o f t h e b i r d s f r o m a l l g r o u p s w e r e a l r e a d y i d e n t i f i e d b y a l u m i n u m l e g b a n d s w h i c h w e r e i n s t a l l e d b y b r e e d e r s a t t h e n e s t l i n g s t a g e . I n c a s e s w h e r e s u c h b a n d s w e r e n o t f i t t e d , p l a s t i c n u m b e r e d b a n d s w e r e p l a c e d o n t h e b i r d s i n E x p e r i m e n t C . T h i s w a s a p r e c a u t i o n t a k e n t o p e r m i t t h e i d e n t i -f i c a t i o n o f p o s s i b l e e s c a p e e s a n d f o r a u t o p s y p u r p o s e s , , s i n c e t h e b i r d s d i d n o t c o m e i n d i r e c t c o n t a c t w i t h e a c h o t h e r f o r t h e s e e x p e r i m e n t s . 63 S o u r c e a n d p r e p a r a t i o n o f t h e I n f e c t i n g i n o c u l u m T w o t y p e s o f i n o c u l u m w e r e u s e d f o r t h e o r a l i n f e c -t i o n o f c a n a r i e s i n E x p e r i m e n t B , a n d o n e t y p e f o r a p i l o t e x -p e r i m e n t a n d E x p e r i m e n t C . T h e s o u r c e i n b o t h c a s e s w a s t h e p o o l o f t i s s u e s f r o m a c a n a r y i n G r o u p I I w h i c h h a d d i e d ( E x p -e r i m e n t A ) . T h e m a t e r i a l w a s c u l t u r e d a n d t h e n s t o r e d a t -20° C . T h e t w o i n o c u l a w e r e : -( a ) A t e n p e r c e n t s u s p e n s i o n i n s a l i n e o f h o m o g e n i z e d p o o l e d t i s s u e w a s p r e p a r e d f o l l o w i n g t h a w i n g , a n d u s e d f o r f o u r ( o n e - h a l f ) o f t h e a r t i f i c i a l l y i n f e c t e d b i r d s i n E x p e r i m e n t B . ( b ) A r e - c o n s t i t u t e d l y o p h i l i s e d c u l t u r e , a f t e r b e i n g c h e c k e d f o r m o t i l i t y w e l l i n a d v a n c e , w a s h e l d a t r o o m t e m p e r a -t u r e i n a t u b e o f R o b e r t s o n ' s M e a t M a s h . T h e d a y b e f o r e t h e e x p e r i m e n t t h e c u l t u r e w a s g r o w n a t 37° C i n a n u m b e r o f t u b e s o f b e e f h e a r t i n f u s i o n b r o t h , s e d i m e n t e d a t U-000 r p m a n d r e -s u s p e n d e d i n a p h o s p h a t e - b u f f e r e d s a l i n e . T h e d e n s i t y w a s s t a n d a r d i s e d t o a t u r b i d i t y a p p r o x i m a t i n g t h a t o f B r o w n ' s n e p h a l o m e t e r t u b e N o . 10. T h i s t y p e o f i n o c u l u m w a s u s e d f o r t h e o t h e r h a l f o f E x p e r i m e n t B , a n d f o r t h e p i l o t e x p e r i m e n t . F o r E x p e r i m e n t C , t h e s a m e p r o c e d u r e a s a b o v e w a s f o l l o w e d , b u t w i t h t h e o r g a n i s m b e i n g s u b j e c t e d t o o n e f u r t h e r c a n a r y p a s -s a g e ( i n t h e p i l o t e x p e r i m e n t ) f r o m w h i c h i t w a s r e c o v e r e d i n p u r e c u l t u r e . 64 This strain was designated "Longworth F n on the basis of i t s original source in Aviary No. 3. Air sampling On several occasions an Anderson a i r sampler was used with blood and MacConkey Agar plates to sample the room air during Experiment B. Results were completely negative for Pasteurella pseudotuberculosis. Prior to obtaining this result, the wearing of face masks was made mandatory for persons enter-ing the room. Cultural procedures on a r t i f i c i a l media (a) From canary tissues in experimental infections A tissue suspension in saline was prepared with a Teflon grinder. This was then plated out on five per cent bovine blood agar and onto MacConkey1s agar. In addition, a few drops of the material were added to a 1 0 . 0 ml tube of Robertson*s Meat Mash (RMM). Following incubation for twenty-four to forty-eight hours at 37° C, typical P. pseudotuberculosis ool-onies on plates were subcultured into both maltose and lactose broth fermentation tubes. If growth occurred only in the RMM then this culture was plated in the same way as the tissue. 65 ( b ) F r o m p r o c e s s e d f a e c e s F o l l o w i n g p r e p a r a t i o n o f t h e p l a t i n g I n o c u l u m ( T a b l e XV)) 0 . 1 m l a m o u n t s w e r e I n o c u l a t e d o n t o t h e s u r f a c e o f M a c C o n k e y a n d b l o o d a g a r p l a t e s : t h e i n o c u l u m b e i n g s p r e a d w i t h a s t e r i l e g l a s s r o d w i t h t h e u s e o f a h a n d t u r n t a b l e . T h e p l a t e s w e r e t h e n i n c u b a t e d a t 37° 0", c h e c k e d t w i c e d a i l y , a n d c o l o n i e s w e r e p i c k e d o f f f o r i d e n t i f i c a t i o n a s a b o v e . I n t h e c a s e o f s u g a r b r o t h s w h i c h s h o w e d f e r m e n t a t i o n o f m a l t o s e w i t h n o g a s , t h e m a l t o s e b r o t h w a s p l a t e d o n t o a s e g m e n t o f a f r e s h b l o o d a g a r p l a t e a n d s t o r e d i n t h e r e f r i g e r a t o r t o a w a i t b i o c h e m i c a l t e s t s t o c h e c k t h e i d e n t i f i c a t i o n . I d e n t i f i c a t i o n o f P a s t e u r e l l a p s e u d o t u b e r c u l o s i s F o r d e t a i l s o f t h e a p p e a r a n c e o f P . p s e u d o t u b e r c u l o s i s , g r o w t h i n p r i m a r y i s o l a t i o n a n d i d e n t i f i c a t i o n m e d i a , a n d f o r b i o c h e m i c a l r e a c t i o n s u s e d , s e e t h e f o r e g o i n g s e c t i o n t i t l e d , • L a b o r a t o r y D i a g n o s i s ' . C a l c u l a t i o n s f o r f a e c a l c o u n t s o f P . p s e u d o t u b e r c u l o s i s a n d f a e c a l d r y m a t t e r o u t p u t ( a ) F o r f a e c a l c o u n t s F o r t h o s e p l a t e c o u n t s w h i c h a p p l i e d t o c o l o n y t y p e s l a t e r c o n f i r m e d a s P . p s e u d o t u b e r c u l o s i s , a f o r m u l a w a s u s e d w i t h t w o v a r i a b l e s a n d t h r e e c o n s t a n t s ( T a b l e X ) . I n t h e t a b l e , t h e c o n s t a n t s a r e u n d e r l i n e d . FACTOR PLATE COUNT (COLONY) X (10 X TOTAL VOL MLS OF DILUENT) (10') X 6/5=TOTAL FAECAL COUNT OF VIABLE ORGANISMS IE 24 - HOUR SAMPLE o H m Total count on plate (average of -fiye small squares) Extra dilution caused by use of 0.1 ml for plating (i.e. 1/10 of dilution unit) This figure in mis represents the degree to which 1/10 total sludge was diluted to 1:50 (W/V) to arrive at a 1:500 dilution (W/V) of faecal sludge These figures give the product of dilution on to plate of l/lO of the total sludge Removal of 1/10 aliquot of sludge by process of standardising to 10 ml vol and removing 1.0 ml Correctional factor to bring 5/6 of faeces (on which count made) to total faeces collected for 24 hours 1XAMPLE 40 X (10 X 12.5)** (10) X 6/5 = 60,000 ** Faecal sludge weight being 0.25. grams TABLE X STANDARD CONVERSION FORMULA EMPLOYED FOR COMPUTING TOTAL EQUIVALENT VIABLE FAECAL COUNT OF PASTEURELLA PSEUDOTUBERCULOSIS PER TOTAL TWENTY-FOUR-HOUR FAECAL SAMPLE OF EXPERIMENTALLY INFECTED CANARIES ON ON 67 Several steps, illustrated by an example, were taken with colony counts to obtain the total equivalent faecal counts. These are shown in the table along with explanations. In the example given here, an original weight'of faecal sludge"*" of O.25 gm was diluted to ten ml in broth. Thereupon, one-tenth of the broth suspension (containing O.O25 gm of sludge) was further diluted to a total volume of 12.5 ml> thereby constituting a 1:500 dilution (W/V) level of the faecal sludge. Plating was carried out with 0 .1 ml of suspension (con-taining 0 .002 gm faecal sludge) which is one-tenth of the unit of dilution. This procedure made a plate from any one bird truly comparable with another, since the same f i n a l dilution (thus the same amount - 0 .002 gm) of faeces was used regardless of actual amount voided by the bird. The three constants give a product factor of 120. In practice, a set of simple tables was prepared which showed the saline volumes required for dilut-ing a l l possible weights of faecal sludge corrected to two dec-imal places. Since the dilution factor was a product of five, the saline volumes came to every whole or half m i l l i l i t r e (actually between g.O and 22 .5 nil). These values in turn were incorporated into a table with a multiplication factor (MF) which was the 1 That portion of 5/6 of original total faecal sample material capable of being sedimented by 10,000 rpm for 15 minutes in an angle centrifuge. 68 p r o d u c t o f each s a l i n e volume and the c o n s t a n t f a c t o r o f 120. I n the example g i v e n , the MF was 15OO. I t remained t h e n o n l y t o a p p l y the a c t u a l ( o r computed) c o l o n y p l a t e count t o the MF t o o b t a i n t h e f i n a l f i g u r e r e q u i r e d . I n the example g i v e n , kQ x 1500 - 60,000. I n many c a s e s , the f i g u r e s were l a r g e . The h i g h e s t output r e c o r d e d on any day ( b i r d No. 16) was c l o s e t o 152 m i l l i o n organisms (I . 5 2 x 10 ) w h i c h was d e l i v e r e d i n a volume of f a e c a l m a t e r i a l w i t h a d r y m a t t e r e q u i v a l e n t of o n l y 0.66 gm. I n t h i s p a r t i c u l a r case the f a e c a l s l u d g e w e i g h t was e x a c t l y h a l f of t h e c a l c u l a t e d f a e c a l DM f o r t h a t b i r d on t h a t day. A t times the f a e c a l s l u d g e ( w h i c h c o n t a i n e d l i t t l e w a t e r a f t e r 10,000 rpm c e n t r i f u g a t i o n ) weighed l e s s t h a n o n e - h a l f of the f a e c a l o u t p u t i n terms o f d r y m a t t e r . I t was not known whe-t h e r most of t h e weight was l o s t i n heavy sediment w h i c h was l e f t on d e c a n t i n g , o r whether more o f i t was l o s t i n s o l u b l e s a l t s and u r a t e s a l o n g w i t h m i c r o - p a r t i c l e s i n t h e d i s c a r d e d s u p e r n a t a n t f o l l o w i n g c e n t r i f u g a t i o n . C e r t a i n l y , t h i s super-n a t a n t c o n t a i n e d v e r y few m i c r o - o r g a n i s m s when t e s t - c u l t u r e d . .(b) C a l c u l a t i o n of the d r y m a t t e r (DM) e q u i v a l e n t weight of the t o t a l d a i l y f a e c a l o u t p u t T h i s has a l r e a d y been d i s c u s s e d t o some degree. The a l i q u o t of o n e - s i x t h of t h e t w e n t y - f o u r h o u r sample of f a e c e s was o b t a i n e d by v o l u m e t r i c p r o p o r t i o n , brought to a i r d r y n e s s a t 37° C o v e r a p e r i o d o f a week or so ( h u m i d i t y of w a l k - i n i n c u b a t o r i s a p p r o x i m a t e l y t h i r t y - s i x p e r c e n t ) and t h e n weighed. 69 The resultant net weight figure is expanded by 6 /5 to obtain that figure which is plotted on the graph chart for each of the inoculated birds. [Execution of Experiments^ Experiment A: Clinico-Pathological and Cultural Observations on Birds Trans-ferred from the Premises of No. 3 Aviary to the Laboratory During an Epizootic Objectives Detailed observations of the course of a canary epizootic of pseudotuberculosis were not available to the author. Such observations appeared to be a fundamental necessity for the evaluation of the data already collected from the No. 1 aviary. In particular, i t was f e l t that the transfer of birds to the laboratory might modify the progress of the disease or yield information to explain the high mortality which had been recorded previously and was already expected for this aviary (No. 3). In retrospect this experiment was of considerable value for comparative purposes in studying the a r t i f i c i a l l y induced infections in Experiment B and Experiment C. 70 Mater ia l s and Methods (a) Source and descr ip t ion of b i rds Two groups of b i rds from aviary No. 3 were used i n t h i s experiment, ( F i g . 7 and Key). Group I : This group consisted of twenty canaries and red-factor canary crosses. They var ied i n plumage colour from pale yellow to orange i n hue, with some possessing dark or white wing feathers. Twelve of these canaries had been picked from F l i g h t " 0 " and various cages where heavy losses or loss of mate had occurred. The remaining eight b i rds were from s ingle cages (four) or were bi rds which flew around loose i n the av ia ry . In addi t ion , four male budgerigars were included from the semi-covered f l i g h t cages at the east end of the b u i l d i n g . S ix canaries i n good health were obtained from the l o c a l zoo for use as contact and i so l a t ed con t ro l s . The l a t t e r source f lock had an excellent health h i s t o r y . Two of these b i rds were maintained i n other premises on the same feed as that used i n the laboratory . Group I I : This group was comprised of f i f t een birds mostly f l edg l i ngs , also obtained from the No. 3 aviary , but co l l ec t ed fourteen days l a t e r when the epizootlc'had been going for s i x weeks and most of the bi rds were dead. Two of the bud-gerigars received with Group I had been housed separately and 71 these were now added to Group II. One of these was then Inoculated orally wl.th,.alcultufe.of P^ pseudotuberculosis. (b) Housing conditions for experimental period (Figs. 12,1:3) The two groups of birds brought were housed in two flights manufactured of slotted angle-steel and welded wire cloth with apertures of one inch by one-half inch. The fl i g h t for Group I measured six feet by four feet by four feet, and that for Group II was slightly smaller. The flights had no bottoms, but were set on a trestle table across which was fed a r o l l of brown double-waxed paper for use as a l i t t e r carrier. Perches were trimmed tree branches which gave a great variation in diameter so that the individual birds could find their most comfortable perch diameter. Experimental Technique General c l i n i c a l observations were recorded, prelimi-nary studies were made to determine the level of excretion of the pathogen into the environment, and those birds which died were studied both culturally and for gross pathological lesions. Four contact control canaries were placed with the f i r s t of two groups of birds after they had been nine days in the laboratory. Figure 12 Experiment A. View of corner of flight cage housing Group I canaries tfromAviary No.3) in the laboratory. Casual observat-ion did not suggest that the birds were sick; however, a dead bird can be noted on the floor and losses were heavy. Figure 13 Experiment A. Close up view of flight shown in Figure 12. Note the tendency of sick birds to huddle in open-type feeders thereby increasing the faecal soilage of seeds. 73 Results and Discussion Cli n i c a l l y the birds were depressed inasmuch as there was no singing and many of them sat in a ruffled attitude on the perches. Practically every bird was active at times however, and they appeared to eat and drink as much as normal birds. When moving or showing interest, the birds uttered hoarse chirps. For a day or two prior to death, birds were usually unable to f l y onto the perches and they would huddle in a corner on the floor. As can be noted from the data (TablesXI&XLt) 75$ of the canaries of Group I, and 55$ of those from Group II were dead by the 77th day following the commencement of the epizoo-t i c . This point corresponds to the reported date of death of the last bird on the premises of aviary No. 3 from whence the birds had been removed. In addition to the frequently occurring yellowish nodular tubercles in spleen and liver, two birds showed the presence of what appeared to be abscesses in the caeca, which are vestigial organs, often termed caecal "buds" in the canary. One bird which showed the most advanced spleen and l i v e r lesions recorded, also showed the presence of pseudotuberculosis in the breast muscles. (Figures;14 and 15). Up to about the 60th day following commencement of the epizootic, isolations of P. pseudotuberculosis could s t i l l be obtained from birds which died with pseudotubercle lesions. THE GROSS PATHOLOGICAL LESIONS RECORDED POST MORTEM ISOLATION OF PPT EXP GROUP SOURCE BIRD IN EXP DAY OF DEATH SINCE SINCE 1ST TOTAL DEATHS SPLEEN LIVER OTHERS ORGANS ISOLATE DESIG. NO. AVIARY TRANSFER LOSSES CUM 7. enlarged tubercles enlarged tubercles description CULTURED SECURED REMARKS GROUP I See 1 f l i g h t •0' 1 31 57. + spleen + sick on arrival Table 2 fl y i n g free 1 31 107. + _ +++small _ liver + Section 5C 3 fl y i n g free 1 31 157. + ++ + ++ spleen + 4 fl i g h t •0' 2 32 207. +++ ++++ +++ _ _ spleen + 5 intact pair 6 37 257. ++ +++ + ++at spleer liver + 20 canaries 6 f l y i n g free 6 37 307. ++ ++ + • t tminute _ spleen + removed bowel + from No.3 7 f l i g h t • 0' 8 38 357. ++ +++ + ++ _ spleen + aviary bowel + 30 days 8 intact pair 8 38 407. •M- +++ + -t-+ _ spleen after 9 single cage 9 39 457. _ _ spleen - NB no gr.lesions beginning liver -of 10 f l y i n g free 12 42 507. +++• ++++ + ++small - spleen + epizootic bowel + 11 f l i g h t • 0' 14 44 557. ++++ ++ _ _ _ spleen + Biochem atypical 12 intact pair 19 49 607. ++++ ++ + ++small catarrhal spleen + Stored in enteritis freezer 9 days 13 intact pair 20 50 657. ++++ +++ +++ ++large emaciated spleen + liver + 14 intact pair 34 64 707. ++ ++ ++ ++ thymus area spleen - NB lesions. No liver - isolate 15 f l y i n g free 36 66 757. ++ ++ ++ ++ _ spleen + Thymus area thymus + independent 16 single cage 53 83 807. _ _ spleen - Tissue pooled 17 single cage 57 87 857. _ _ _ _ l i v e r & spleen -18 single cage 70 100 907. _ - liver & Tissue pooled spleen - Tissue pooled 19 single cage 70 100 957. _ _ _ _ liver & spleen -1 The surviving canary from Group I was later included in experiment ."J)?' This bird, along with the survivors of Group II ultimately died from general d e b i l i t y . 2 Two of four male budgerigars were received from the aviary on the same date as the canaries. Two of these birds were placed with Group I as contact controls. They remained healthy for the 61-day observation period. When autopsied later, no pathological lesions were seen except for hypertrophy of the thyroids. TABLE XlQ RECORD OF MORTALITIES, PATHOLOGICAL LESIONS, AND ISOLATIONS OF PASTEURELLA PSEUDOTUBERCULOSIS FROM iCANARIES TRANSFERRED TO LABORATORY FROM AN AVIARY DURING THE COURSE OF AN EPIZOOTIC (GROUP I TRANSFERRED 30 DAYS AFTER LOSSES COMMENCED) •S3 6XP GROUP DESIG. SOURCE BIRD IN NO. AVIARY EXP DAY OF DEATH TOTAL THE GROSS PATHOLOGICAL LESIONS RECORDED POST MORTEM ISOLATION OF PPT SINCE SINCE 1ST DEATHS SPLEEN LIVER OTHER ORGANS ISOLATE TRANSFER LOSSES CUM % enlarged tubercles enlarged tubercles description CULTURED SECURED REMARKS GROUP II See Table Section 5C 15 canaries removed from No.3 aviary 14 days following remova1 of Group I Table 1 Exact 1 45 6.6% ++ ++ _ _ spleen + 2 sources not 1 45 13.37. ++ ++ - - caecal absc. peritonitis spleen bowel + 3 known 3 47 19.87. M M +++ + _ caecal absc. spleen + very large bowel + 4 5 49 26.47. ++++ +++ + ++ ++large spleen + Mostly li v e r + 5 fledglings 8 52 '33.07. ++++ +++ + + ++small spleen 6 from a l l over 16 60 39.6% M M +-M- ++++ large breast musc.tuberc. pool + 7 16 60 46.2% - - - very poor flesh pool -8 33 77 54.87. +++ _ -M-Small emaciated pool -for inoc CONTROLS Four of six contact control canaries placed in fl i g h t 9 days after Group I N/A N/A N/A N/A 21 N/A 257. 22K N/A 50% 50 N/A 757. 61 N/A 100% emaciated pool emaciated pool emaciated pool emaciated pool 1 The seven surviving canaries from Group II were later included in experiment g as potentially infected. As noted ~H a l l of these birds eventually succumbed from general debility. — • — ' 2 The remaining two of four male budgerigars (footnote 2, TableXlO were placed with Groun II canaries, and one bird, the 'infected control' was dosed orally with 0.5 ml of a broth culture of Pasteurella pseudotuberculosis. This bird died with c l a s s i c a l lesions of Pseudotuberculosis twelve days post inoculation. The other bird remained healthy during the obervation period. 3 The two remaining control canaries were u t i l i z e d as isolation controls. ( H S« TABLE XII RECORD OF MORTALITIES, PATHOLOGICAL LESIONS, AND ISOLATIONS OF PASTEURELLA PSEUDOTUBERCULOSIS FRCM CANARIES TRANSFERRED TO LABORATORY FROM AN AVIARY DURING THE COURSE OF AN EPIZOOTIC (GROUP II TRANSFERRED 14 DAYS AFTER GROUP I, I.E. 44 DAYS AFTER LOSSES HAD COMMENCED) ->3 Figure 14- Experiment A. Canary from Group I. Spleen and liver lesions at autopsy (spontaneous infection). The spleen shows as a dark cylindrical organ. The liver l i e s either side of the spleen and below the gizzard in this picture.The kidneys are in the background to the right. Pseudo-tubercles were not seen In renal tissues. Figure 15 Experiment A. Canary from Group II. Breast muscle and l i v e r lesions at autopsy of a spontaneous infection case. The latter may be seen through the thin, unopened abdominal wall. (caudal to sternum) The affected tissues from this bird were stored frozen and later used to infect birds experimentally (Experiment B), by oral inoculation. Figure 15 77 Following this point, six more birds died by the 100th day. Of these, one only showed discrete pseudotubercles in the l i v e r , and this bird plus one other showed splenic enlargement. No isolation of P. pseudotuberculosis could be obtained from these six birds or any of the remaining eight which died without typical cross lesions in a succeeding experiment at points ranging between the 102nd and 241st day from the start of the epizootic. (Experiment B). The four contact control birds sick-ened and died, one with an enlarged spleen. No isolations of P. pseudotuberculosis were obtained. Two additional isolation control birds remained healthy on the same feed at the author's residence. Conclusions Deaths continued at a high rate in the groups under observation with $0% of Group I being dead by twelve days follow-ing the transfer, and 46$ of Group II being dead in sixteen days following the move. It appears from this and from the compar-ison already drawn between the events at the aviary and those in the laboratory, that the disease was not significantly modi-fled in i t s general course by the transfer. • Clinically, there did not appear to be any feature of diagnostic significance which was observed in the affected canaries under close observation. From the pathological and cultural data, one could deduce that this infection tends to 7 8 b u r n i t s e l f o ut e v e n t u a l l y , b e f o r e a l l t h e b i r d s are dead; b u t t h a t t h e deaths c o n t i n u e due t o g e n e r a l d e b i l i t a t i o n even a f t e r the b i r d has e l i m i n a t e d the v i a b l e pathogens f r o m i t s system. Two p o s s i b l e f a c t o r s a r i s e here w h i c h c o u l d c o n t r i b u t e t o t h i s e f f e c t . One i s the p r o d u c t i o n of b a c t e r i a l e n d o t o x i n d u r i n g the r e s o l u t i o n o f p s e u d o t u b e r c l e s , and the o t h e r i s the i n c r e a s e d d i f f i c u l t y w h i c h weakened c a n a r i e s have i n c r a c k i n g t h e i r seeds so as t o m a i n t a i n the i n t a k e of d i g e s t i b l e n u t r i e n t s . I t seems l i k e l y t h a t the second f a c t o r c o n t r i b u t e d t o the u l t i -mate d e a t h o f b i r d s w h i c h s u r v i v e d beyond one hundred d a y s , s i n c e t h e y were em a c i a t e d a t d e a t h . Undoubtedly th e more c o m f o r t a b l e environment of t h e l a b o r a t o r y extended the l i f e o f s uch d e b i -l i t a t e d b i r d s beyond t h a t p e r i o d e x p e c t e d i n t h i s p a r t i c u l a r a v i a r y . P r o b a b l y t h e most s i g n i f i c a n t f i n d i n g was t h a t of two b i r d s w i t h a b s c e s s e s i n t h e c a e c a l buds. S i n c e b o t h t h e s e b i r d s were h e l d i n the f r e e z e r a w a i t i n g a u t opsy and c u l t u r e , t h e r e was no h i s t o l o g i c a l s t u d y made o f t h e s e p a r t i c u l a r l e s i o n s . T h i s f i n d i n g w i t h i t s p o s s i b l e i m p l i c a t i o n o f heavy f a e c a l d i s s e m i n a t i o n s u g g e s t s i t s e l f as a p r o b a b l e e x p l a n a t i o n f o r the a p p a r e n t l y v i g o r o u s r o l e o f the c a n a r y i n s p r e a d i n g i n f e c t i o n t o o t h e r members of t h e a v i a r y . I n a l l c a s e s w i t h t y p i c a l g r o s s l e s i o n s where bowel c u l t u r i n g was c a r r i e d o u t , an i s o l a t e of P. p s e u d o t u b e r c u l o s i s was o b t a i n e d . The f o u r c o n t a c t c o n t r o l b i r d s s i c k e n e d about 79 nineteen days after entry to the flight, and died between the 21st and 6 l s t day of the experiment. Of these, three died and one was k i l l e d . It is f e l t that no conclusions of any kind can be drawn from these deaths of control birds in the absence of pseudotubercle lesions. Three budgerigars remained healthy. One was inoculated orally with broth cul-tures and died twelve days post-inoculation with typical lesions and cultural recovery of ps eudotube rculos i s . Experiment B: Contact Infection Experiment with Healthy and A r t i f i c i a l l y -Infected Birds Objectives A tentative f i r s t attempt to demonstrate contact infection between naturally infected and healthy canaries had been carried out in Experiment A. The four healthy birds sickened and one was k i l l e d and three died. There was a d e b i l i -tating process which was absent in their two mates maintained on the same feed at other premises. P_j_ pseudotuberculosis could not be Isolated when the birds were examined at autopsy. However, this did not rule out the possibility of infection having taken place during the f i r s t two weeks or so. In light of this and the fact that the healthy canaries had 80 not been i n t r o d u c e d t o the i n f e c t e d group u n t i l so l a t e i n the c o u r s e o f t h e e p i z o o t i c , i t was d e c i d e d t h a t an experiment s h o u l d be set up s p e c i f i c a l l y d e s i g n e d f o r t h i s p u rpose. I n b o t h No. 1 and No. 3 a v i a r i e s , the b u d g e r i g a r s had remained c l i n i c a l l y f r e e o f p s e u d o t u b e r c u l o s i s . S i n c e i n the n a t u r a l s t a t e , the b u d g e r i g a r ( a p s i t t a c i n e ) i s a top f e e d e r among s e e d - b e a r i n g p l a n t s , whereas the c a n a r y ( a p a s s e r i n e ) i s a ground f e e d e r , the q u e s t i o n a r o s e as t o whether t h e c a n a r y w o u l d be l e s s l i k e l y to become i n f e c -t e d i f i t was not a l l o w e d a c c e s s t o the c a e o a l l y c o n t a m i n a t e d f l o o r environment of the cage. F o r t h e purpose of t e s t i n g t h i s t h e o r y , a double cage was d e s i g n e d i n c o r p o r a t i n g two extreme l e v e l s of s a n i t a t i o n . M a t e r i a l s and Methods ( a ) e x p e r i m e n t a l b i r d s S i x t e e n h e a l t h y c a n a r i e s were o b t a i n e d f r o m a h e a l t h y f l o c k w h i c h had been c l o s e d f o r s e v e r a l y e a r s , and f r e e o f e p i z o o t i c s d u r i n g t h a t p e r i o d . (b) h o u s i n g F o r t h i s experiment a cage o f s l o t t e d a n g l e s t e e l and w i r e c l o t h was made w i t h o v e r a l l d i m e n s i o n s of two f e e t 81 by four feet by two feet . This was d iv ided i n hal f by a par-t i t i o n of the same welded wire c l o t h , and one one side there was a fa l se f l o o r , b u i l t up four inches from the cage bottom, constructed also of wire c l o t h , one inch by one-half inch . Experimental Technique Two groups of th i r teen canaries were assembled, each sub-divided in to two groups of four and one group of f i v e . These sub-groups consisted of f ive healthy canaries obtained from an aviary i n B r i t i s h Columbia (the remaining b i rds i n th i s aviary served as ' o f f experiment 1 con t ro l s ) ; three more b i rds from th is source; one of the i s o l a t i o n cont ro l canaries from Experiment B ( f reshly brought on premises) which were to be introduced and infected; and four of the p o t e n t i a l l y in fec -ted, or perhaps immune, survivors of the No. 3 aviary ep i zoo t i c . The group held i n the l e f t hand side of the double cage were allowed access to the f loor and were given f l oo r feed and water receptacles which soon became heavi ly contains inated wi th faeces. This f l i g h t was designated 'standard* cage. The r ight hand f l i g h t contained a wire fa lse bottom and the feed, water, and g r i t containers, as w e l l as the cu t t l e bone were a l l suspended on the side wire as close as possible to the top of the cage. In addi t ion to t h i s , the wire f loor and wal l s of the r igh t hand f l i g h t were cleaned of a l l faecal 82 mater ia l once d a i l y by use of a ce l l u lo se sponge and Roccal 5 0 0 PPM i n warm water. The water fountains were flushed out and r e - f i l l e d d a i l y . This f l i g h t was designated ' t e s t cage' . The sub-groups of f ive healthy and four p o t e n t i a l l y infec ted (PI) b i rds were placed i n the cage so as to t o t a l nine bi rds i n each s ide. Af te r eighteen days, eight more birds were introduced to the experimental room, infected wi th 0.1 ml of a reconst i tu ted broth cu l ture of the Longworth F s t r a in of P. pseudotuberculosis by means of an 0.2 ml p ipe t t e r , and placed i n the cages. Results The resu l t s w i l l be presented i n summary only since i t i s f e l t that the experiment was not successful , due i n part to improper design. The eight birds which were inoculated o r a l l y became infec ted and seven d ied . With one or two exceptions (Fig.16) they showed c l a s s i c a l lesions of pseudotuberculosis. The poten-t i a l l y infected bi rds continued to d ie as described i n Experiment B, and eventually a l l succumbed to a d e b i l i t a t i n g disease not recognizable as pseudotuberculosis. The healthy birds a l l became s i ck and nine of the ten eventually d ied . These also suffered from a d e b i l i t a t i n g disease not recognizable as pseudotuberculosis. Figure 16 Experiment B. Gross view at autopsy of thymus lesion in a canary which died 34 days following oral inoculation .with Pasteurella pseudotuberculosis. Previously, isolations were obtained on two occasions from smaller lesions in the thymus area in birds spontan-eously infected (Aviary No. 3) . This bird also showed extensive lesions in lungs and abdominal viscera. . Figure 16. 84 Conclus ions U s e f u l d a t a were secured on the a b i l i t y to a r t i f i -c i a l l y i n f e c t c a n a r i e s . One i s o l a t i o n of P. p seudotubercu los i s was o b t a i n e d from the f a e c a l droppings from the s t andard cage by means of i n o c u l a t i o n of sediment i n t o a guinea p i g . Thus i t can be s t a t e d tha t the h e a l t h y c a n a r i e s were exposed to an i n f e c t e d environment. The h e a l t h y c a n a r i e s e v i d e n t l y s u f f e r e d from a d e b i l i t a t i n g d i sease which might have been t r a n s m i s s a b l e ; however, I t was f e l t t h a t t h e i r deaths were more d i r e c t l y due to n u t r i t i o n a l d e f i c i e n c y . I n f o l l o w i n g exper iments , a b e t t e r m i x t u r e of seeds was employed to enable s i c k b i r d s to p i c k those seeds w i t h h u l l s more e a s i l y cracked than those of the p l a i n canary seed. Experiment C; Experiment w i t h Quant i t a t ive Determina t ion of Faeca l D i s -seminat ion O b j e c t i v e s (a) To reproduce p seudotubercu lo s i s by o r a l i n o c u l a t i o n of canar ie s w i t h a r e s u l t a n t d i sease course and p a t h o l o g i c a l l e s i o n s s i m i l a r to those recorded d u r i n g the course o f e p i z o o t i c s of the spontaneous d i sease i n B r i t i s h Columbia . 85 (b) To measure, q u a n t i t a t i v e l y i f p o s s i b l e , the f a e c a l d i s s e m i n a t i o n l e v e l of P. pseudotuberculosis achieved by the. i n o c u l a t e d b i r d s from the day f o l l o w i n g i n o c u l a t i o n to the day of death; or, i n the event of recovery, the day of s a c r i f i c e . (c) To shed f u r t h e r l i g h t on the probable r o l e of the oanary i n the e p l z o o t i o l o g y of spontaneous disease i n i t s own s p e c i e s , and to a l l o w an estimate to be made of i t s pos-s i b l e r o l e i n zoonotic pseudotuberculosis. M a t e r i a l s and Methods (a) experimental b i r d s K T h i r t y b i r d s were r e c e i v e d from a d e a l e r i n Van-couver. These were ordered from a c l o s e d - f l o c k a v i a r y i n B r i t i s h Columbia possessing an e x c e l l e n t h e a l t h record. The d i s t r i b u t i o n of sex was s i x males and twenty-four females. Weights ranged from f i f t e e n to t h i r t y grams. Their breeding d i d not in v o l v e any h y b r i d s , however the col o u r and wing mark-ings of the canaries v a r i e d considerably. I t should be noted that at no time have any observations made by t h i s author on spontaneous or experimental pseudotuberculosis suggested that there i s any sex d i f f e r e n t i a l i n s u s c e p t i b i l i t y of canaries to P. pseudotuberculosis i n f e c t i o n . The b i r d s used f o r t h i s experiment were maintained at the l a b o r a t o r y i n good h e a l t h f o r a p e r i o d of one month before the experiment commenced. Figure 17 Experiment C. View of the canary-battery unit which was used for the quantitative fecal dissemin-ation experiments. The operator removing hook-on seed and cuttle containers from the wire fronts of the cubicles. This was in preparation for the evening collection of twenty-four hour faecal samples. Figure IS Experiment 0. Another view of the canary battery. Cleaned and sterilized metal floor trays with wire grids are replaced following faecal collection. Figure IS Figure 1 9 Experiment C. Further view of canary battery. This picture shows fountain-type grit and water containers, hook-on feeders,and spring c l i p wooden perches. The perches were replaced with sterile substitutes daily,following faecal sample collections. A l l wooden and metal surfaces received a thin coat of molten parawax. Figure 20 Experiment 0. Canary battery showing close up of individual cubicle. 87 . F i g u r e 20 BIRD NO. CLINICAL APPEARANCE DEATH TIME DAYS PI AUTOPSY LESIONS ATTEMPTED CULTURAL RECOVERY EAECAL CULTURES • - . Tissue "Result 1 Perky to the end 30 Enlarged spleen air sacs, yellow liver kidney spleen air sac bowel +, .+ + not attempted initially 2 Non clinical recovered 70 sacrificed None. Excellent condition Kidney liver spleen bowel - not attempted initially 3 Ruffled after 3 days . 12 Very sick after 6 days Typical nodules spleen and liyer of p. pseudo Kidney spleen liver bowel • . + + not attempted initially 4 Normal and perky Death sudden 57 Right lung caseous mass left air sacs caseous material spleen kidney liver right lung cloaca + not attempted initially 5 Rough on 7th day 8 Spleen enlarged 2 X. No other spleen lung livery rt kid bowel + + + 4th day pos. (few cols) 5th & 6th.days negative 6 Rough and depressed on 5"th day 10 Both lungs and air spleen sacs caseous materia liver al spleen enlarged lung with one tubercle air sacs bowfil + + + + -f-day.death sample pos. 5"th 6th, 7th days negative TABLE X I I I EEEECT OE INOCULATION OE BROTH CULTURE ORALLY IN SIX CANARIES - STRAIN "L0NGW0RTH E". INOCULUM CONSISTED OE 0.2 ML BROWN #10 EQUIVALENT RESUSPENSION 0E TWENTY-EOUR-HOUR GROWTH"AT 37° C co CO 89 (b) Housing For these experiments, a battery of twenty-four cubic les was constructed of wood and wire (F ig s . 1? - 2b). The dimensions of the i n d i v i d u a l cubicles were eight inches by twelve Inches by eight inches. (A f u l l descr ip t ion of the const ruct ion w i l l be found in the Appendix.) Experimental Approach Fol lowing the completion of Experiment B, a ser ies of pre l iminary experiments were ca r r ied out to obtain guide l i n e s i n materials and methods for Experiment C. The pre-l iminary work included a p i l o t experiment which has been pre-v i o u s l y referred to, wherein s i x bi rds housed i n the s p e c i a l l y b u i l t cubic le battery were Infected o r a l l y . The resu l t s i n terms of pathology and recovery of the organisms from t issues and faeces are to be found i n Table XIII. A number of conclusions were made from the p r e l i m i -nary work which may be l i s t e d under the fo l lowing headings. (a) Effectiveness of the Inoculum There was every reason to fee l that the s t r a i n and dosage selected was sa t i s fac tory to in fec t the b i rd s , and to produce c l a s s i c a l les ions of pseudotuberculosis. A t o t a l of ten bi rds had been inoculated wi th broth 90 cultures of the Longworth F . s t r a i n i n Experiment B, and the preliminary p i l o t experiment. Of ten b l r d 6 inoculated, one only recovered and seven died within seventeen days of inoc-u l a t i o n . Two died at extended times l a t e r . A t o t a l volume of 0 .2 ml was used i n the p i l o t pre-liminary experiment. This was f e l t to he too great f o r a s i n -gle dose into the esophagus since birds regurgitated and suf-ered a b r i e f collapse from anoxia, and the resultant lesions i n the lung suggested that culture material had been inspired into the bronchial system. A s i m i l a r volume (0 .225 ml) was then used in Experiment 0, but was s p l i t into three d a l l y doses of 0.075 each. In administering the inoculum, a serology pipette of 0 .2 ml capacity was discontinued i n favour of a s t a i n l e s s s t e e l teat cannula. (b) C o l l e c t i o n and storage of the faeces Collection methods were changed i n several respects. The use of waxed paper drop sheets was discontinued i n favour of coating the metal of the drop tray with a thin layer of molten wax. For the p i l o t experiment the faeces were stored at 5 ° C f o r days or weeks p r i o r to c u l t u r i n g . Recoveries of P. pseudotuberculosis from the faeces of birds No. 5 and No. 6 of the p i l o t group (the f i r s t two to die) were quite inconsistent. Since storage at 5 ° C introduced an unknown 91 factor*, i t was decided to cut this delay to less than twenty-four hours, except for two days during the experiment when i t was necessary to sharp freeze the material and process i t later. (c) Culture of faecal material It was found that 0.1 ml of a 1:500 dilution of faecal sludge would yield a bacterial colony concentration suitable for counting with a Quebec Colony Counter ohJa.MacConkey agar plate. For qualitative work, blood agar plates were also used, containing five per cent bovine red c e l l s . (See Table XV). In selecting the secondary isolation media, i t was noted that the best growth with uniform turbidity occurred with beef heart infusion broth; however, maltose peptone water tubes (with gas tube) were quite satisfactory for supporting growth of P. pseudotuberculosis. Furthermore, other members of the oanary bowel flora, possessing similar colony characteristics did not apparently have the a b i l i t y to ferment maltose without gas production. Experimental Technique The complex series of steps involved in this exper-1 It has recently been shown that P. pseudotuberculosis w i l l multiply in certain media at this temperature refrigera-tion (Paterson and Cook, 1962). 92 lment is presented as a flow diagram in Tables XIV and XV. The experimental work was made up of a number of phases which can be l i s t e d as follows: (a) Acclimatisation phase The birds were placed in the cubicles about one week before the experiment started, following thorough preparation of the surfaces. (b) Inooulatlon phase After collection of one pre-inoculation set of twenty-four hour faecal samples, the birds were inoculated daily over a three day period with 0 .075 m l o f a turbid sus-pension of P. pseudotuberculosis strain "Longworth F". (c) Faecal collection phase Each evening a complete set of twenty-four hour faecal samples was collected aseptioally from the twenty Inoculated and four control birds. The technique appears in Table XIV. (d) Faecal processing and culture phase The steps outlined on the accompanying flow 93 TABLE XIV ASEPTIC TECHNIQUE FOR COLLECTION•OF 24-HOUR FAECAL SAMPLES FROM CANARIES 1. Prepared 24 sterile, S/C, pre-labelled glass vials in  wooden "block j ! 2. Removed a l l feed and water containers from wire fronts  of cubicles to prevent possible cross contamination  during collection procedures 3. Faecal drop trays removed from six cubicle cages at a  Time and a plastic baffle was hung in place to prevent  escape of canaries through apertures" 4 . Faecal material adhering to grid over drop tray was  Teased loose with sterile tongue depressor, this f e l l  onto drop tray ready for collection I 5. Continued action with the tongue depressor loosened THe faecal material from the drop tray, moved i t to  one corner and guided i t into the collection v i a l  corresponding to the cubicle number for the canary 6. While action No. 5 continued, the cleaned-off drop trays  and grids were boiled for three minutes in an Instru- ment~steriliser which had a thin layer of molten wax on the surface of the boiling water I 7. Upon removal from the s t e r i l i s e r , each grid and tray  was wiped with a sterile paper towel. This l e f t a ~ TKin layer of wax upon the sterile metal surfaces I S>. While the trays and grids were out of the cubicles, the  wire fronts were wiped with sterile paper towels dam- pened In cold tap water j 9. Immediately following action No. g, the c l i p perches  were removed from the cages and placed in an instru- ment s t e r i l i s e r . A spare set of ste r i l i s e d , waxed,  and dried perches was replaced dally II 10. The sterile trays and grids were replaced in the cub- Teles in sets of six. The thin wax layer prevented  close adhesion of faeoal material to the metal surface  TEereby making the above operation fast and efficient 94 11. A l l water fountains were emptied and rinsed thoroughly  wTth cold running water. The feeder dishes were tip- ped out completely and h a l f - f i l l e d with fresh seed. T5e cuttle was replaced in the same cup, or discarded i f i t had been thrown out "by the bird 12. The plastic baffle which projected from between the decks 1 to protect the lower feed cups from contaminat- ion) was removed and washed thoroughly under running"water 13* Freshly collected sets of 24-hour faecal samples removed  to 5" C storage overnight : 95 TABLE NO. XV PROCESSING AND CULTURAL PROCEDURES USED IN OBTAINING FAECAL COUNTS AND FAECAL DRY MATTER OUTPUT ON A DAILY BASIS INDIVIDUAL CANARY battery with cubicles was completely serviced at 20:00 hours daily QUANTITATIVE CULTURING Complete 24 hour faecal sample  removed from drop tray to screw cap IS/CJ v i a l overnight at 5° C 10 ml SPSS added to soften  faecal material in S/C using  Cornwall Syringe" stand 3 ° mins at R/T V i a l shaken vigorously for 30  sees to homogenize faecal material in S/c v i a l groups of four samples Tipped through stainless steel  sieve to beaker. Vial rinsed and sieve flushed with total of 18.0 ml sterile saline solution physiologi cal QUALITATIVE CULTURING discontinued after f i r s t ten days as found to be unnecessary One fresh canary dropping  collected on waxed paper  Inserted when drop tray  removed for cleaning With use_4 mm platinum  loop plate out on Blood Agar Plate (BAP) MacConkey Agar Plate (MacCAP) incubate 24 hrs at 3 7 ° 0 Check plates for colonies  suggestive of being Pas- teurella pseudotuberol. Beaker contents returned to  3/0. V i a l s and topped up to ' grad mark or 3 0 ml with SPSS' Shake again and allow  to stand f o r 1 0 minu- tes to allow s e t t l i n g  heavy sediment One 5.0  ml a l i - quot Decant to stainless s t e e l  centrifuge tube of known"" weight Centrifuge 10.000 RPM/15  mins In angle centrifuge Invert to discard super-natant drain f o r 30 seconds Weigh tubes on analy- t i c a l balance and subtract  tube weight to give net weight damp sludge Added nutrient broth to  give 10 ml W/V t o t a l  1UU 3 . 0 mm glass beads Pick such colonies o f f  l a t e to maltose broth  ubes with gas tube ~ f Incubate to 72 hours at 3 7 " C and check f o r acid  no gas Maltose fermenters Gas  (no gas; and non- produ-fermenters cers 1 Discard Streak on segment  of blood agar  plate Incubate ^7° 0 for 2k hours Store at 5° C f Biochemical I d e n t i f i c a t i o n check ~~ " agitated f o r uniform re-suspension 97 Removed 1 .0 ml (l/lO of  resuspension sludge) to  saline blank so as to"glve  1:50 W/V dilution of suspension shake thoroughly Pipetted 0 . 1 ml onto surface of MacCAP and Removed 6 . 0 ml to 12 x  2>5 mm plastic v i a l (cork  stoppered) labelled -20° C deep freeze for,  use in emergency"* At time of plating a spread with sterile glass  rod using turntable incubate 37° 0 Examined 2^ hours, at 48 hours (most  useful &. bb-7° hours Suspicious colonies  described and counted on Quebec Colony Counter Picked off representative numbers to maltose broth tubes 37 C incu-bation to 72 hours broth culture of pseudo TB~ streaked Control MaoConkey agar  plate examined for colony  size. If negative for  suspicious colony Discard Acid formation  No gas Growth with Acid if Gas no acid form Plate on BAP Further inou- Discard batlon at 17° C Storage 5° C 98 DETERMINATION OF FAECAL DRY MATTER P l a s t i c P e t r i d i s h dishes grouped on t r a y s 3 7 ° C Incubator  Covered to kee"p out  dust and allowed to  dry f o r 7 days Weighed on a n a l y t i c a l balance C a l c u l a t i o n x 6 to g i v e  t o t a l f a e c a l dry matter  output""'f or 2*4- hour p e r i o d f o r i n d i v i d u a l b i r d 99 chart (Table XV) took up much of the experimental time daily. However, in retrospect i t was f e l t that the results Justified this concentration of effort. (e) Examination of cultures In the early morning and late afternoon, a l l primary isolation plates were checked. Colony types were ex-amined, selected, described, counted, and picked off into maltose broth tubes (see flow chart Table XV). Other tubes of maltose broth, already Incubating, were examined daily for acid production with no gas. Tubes showing such character-i s t i c s , (or growth with no fermentation), were streaked out with a loop into blood agar plates for incubation and storage. (f) Pathological examination As birds died, they were chilled, autopsied, and cultured. Gross pathological examinations were carried out, and tissues were fixed. Surviving birds, a l l of which had ceased to shed viable P. pseudotuberculosis were autopsied and cultured on the 21st day after the conclusion of the course of inoculations, i.e. the 24th experimental day. (g) Biochemical identification of isolations Representative numbers of faecal isolates of 100 presumed P. pseudotuberculosis were checked biochemically for each bird following termination of the experiment. Checks were made of a l l f i r s t counts, peak counts, and f i n a l counts for individual shedder birds. Results and Discussion (a) Clinical appearance of the birds Within about three days of completion of the series of oral inoculations, i.e. around the sixth experimental day, the birds appeared subdued. There was l i t t l e or no singing from the Inoculated birds. Two of those birds which subsequ-ently died (No. 3 and No. 17) were noticed to be ruffled from this point onward. On some days they appeared to be improved. The activity of the birds was, of course, limited by the cub-iole size and they did not have the stimulus of being able to see each other. One bird, No. 17, seemed to be particularly agitated as though constantly hungry and i t emptied the feed dish daily. Most of this feed went on the floor of the room. It was interesting that this bird never showed a positive faecal count until the fifteenth experimental day which was eight days before death. Another bird, No. 12, was also ruf-fled towards the end of the experiment. It l a i d two eggs on the 11th and l 4 t h experimental days, but according to the faecal count and autopsy findings, i t reached the recovery point on the 20th experimental day. Bird No. 1 sat a great deal on the 1 0 1 wire toward the end of the experiment. A number of the b i r d s s u f f e r e d from wet s t o o l s t r a n s i e n t l y . Three of these were non-i n o c u l a t e d c o n t r o l b i r d s . A lactose-fermenting mucoid organ-ism appeared on the p l a t e s . This o o n d i t i o n was n o t i c e d to be un r e l a t e d to the c l i n i c a l s i g n s of depression or of m o r t a l i t y due to the t e s t organism. As the experiment progressed, the c o n d i t i o n c l e a r e d up. The causative organism was assumed to be a low grade pathogen, the spread of which commenced before the b i r d s were separated and was checked by the i s o l a t i o n of the b i r d s from each other. As f a r as the i n f l u e n c e on the f a e c a l count of P a s t e u r e l l a was concerned, i t was n o t i c e d t h a t t h i r t e e n of the i n o c u l a t e d b i r d s never showed s o f t s t o o l s d u r i n g the whole course of the experiment. Of t h i s group, two d i e d , seven a d d i t i o n a l b i r d s gave a p o s i t i v e count, and f o u r of the b i r d s never showed a p o s i t i v e count. Doubtless i t would not have been n o t i c e d i f the f a e c a l samples had not been c o l l e c t e d . One b i r d , No. 13, was seen to be wheezing on the l g t h experimental day. This was the same day that the b i r d reached the po i n t of recovery. The d i s t r e s s may have been asthmatic, or due to endotoxin formation as the b a c t e r i a l c e l l s were broken down i n the r e t i c u l o - e n d o t h e l l a l system. (b) M o r t a l i t y As s t a t e d elsewhere the m o r t a l i t y r a t e among inoc -u l a t e d b i r d s was twenty per cent. This was lower than expected, 102 but i n view of the large number of birds which*showed p o s i t i v e f a e c a l counts (80 per cent) the lower.mortality may have r e s u l -ted i n the data being of greater s i g n i f i c a n c e . A l l the birds which died showed lesions and yielded a culture of P. pseudo- tuberculosis. (c) ^Variance i n faecal excretion and weight of birds There were several reasons f o r t h i s measurement.being taken and recorded on the graph charts f o r each b i r d . F i r s t , It allowed d i r e c t c o r r e l a t i o n to be made between f a e c a l colony count and f a e c a l volume i n terms of dry matter. This was desirable where marked fluctuations of high peaks are noted on the graph. Secondly, the r e l a t i v e food intake of the b i r d could be measured i n no other way. As i t turned out, the birds did not cease to eat, although some varied t h e i r Intake shar-ply. Any r e l a t i o n s h i p between peaks of faecal count, and appetite, or excretory a c t i v i t y of the bowel could be noted on the graph chart. Thettotal food intake could be roughly calculated f o r each b i r d during the course of the experiment, and correlated with weight v a r i a t i o n which occurred during the twenty-five experimental days or u n t i l death. Considering weight variations i n retrospect there was an unfortunate omission i n that the weight of the non-inoculated controls was not taken on the f i r s t experimental day. Actually the main function planned for the control group 103 was to provide b a c t e r i o l o g i c a l evidence p a r t i c u l a r l y of whether the l i v e organism contained i n the inoculum was capable of spreading from one canary to another, and more important, from one s l i d e t r a y to another. This purpose was achieved w e l l enough. In a d d i t i o n there were four i n o c u l a t e d b i r d s which never passed the organism i n the faeces. This group i s of g r e a t e r comparative value i n some respects than the non-i n o c u l a t e d c o n t r o l s could have been. Of t h i s group two gained s l i g h t l y i n weight, one l o s t s l i g h t l y , and one h e a v i l y . Of the group which showed f a e c a l counts a l l showed a weight l o s s v a r y i n g from about $.7% up to 40.6$. These observations are recorded i n Table XVII which summarizes much of the data. As u n c o n t r o l l e d c l i n i c a l observations, the l o s s i n weight of a l l the shedders, but of only two of four non-shedders, would appear to be d e f i n i t e l y a s s o c i a t e d w i t h the experimental d i s -ease. The very magnitude of the weight l o s s e s suggests that the environmental and n u t r i t i o n a l f a c t o r s could not have been s o l e l y r e s p o n s i b l e . Since most a u t h o r i t i e s describe v a r y i n g degrees of l o s s of c o n d i t i o n f o r n a t u r a l l y o c c u r r i n g pseudo-t u b e r c u l o s i s , the f i g u r e s are not s u r p r i s i n g . (d) Presence and amplitude of f a e c a l counts f o r v i a b l e P a s t e u r e l l a pseudotuberculosis The data c o l l e c t e d from t e s t i n g of f a e c a l m a t e r i a l , and p a t h o l o g i c a l examination accompanied by b a c t e r i o l o g i c a l 1 0 4 c u l t u r i n g , allowed a f a i r assessment to be made of the course and pathology of experimental pseudotuberculosis i n ca n a r i e s . The main p o s t u l a t e f o r t h i s experiment was based on the ex-pected s i g n i f i c a n t l e v e l of shedding of v i a b l e P. pseudotuber- c u l o s i s organisms i n the canary faeces f o l l o w i n g t h e i r inocu-l a t i o n per orurn. The experimental group, t o t a l l i n g 24 b i r d s , f a l l s i n t o three groups based on the presence or otherwise of v i a b l e shed organisms. The f i r s t of these, group A, c o n s i s t i n g of 16 b i r d s , a l l shedders, can be f u r t h e r d i v i d e d i n t o three sub-groups designated A - l to A - 3 , based on v a r i a t i o n s i n the p a t t e r n of output of v i a b l e organisms i n the d a l l y f a e c a l e x c r e t i o n s . (Table XVI). A more complete summary of data c o l l e c t e d on i n d i v i d u a l b i r d s appears i n Table XVII, covering a l l b i r d s which r e c e i v e d the i n f e c t i n g inoculum. From the above i t can be noted that the p o s t u l a t e i n v o l v i n g f a e c a l shedding was f u l f i l l e d , since s i x t e e n (SO per cent) of the twenty i n o c u l a t e d b i r d s were shedders, many of them on a tremendous s c a l e ( F i g u r e s 21-4o), even some which l a t e r recovered (as Judged by c e s s a t i o n of shedding and negative c u l t u r e upon autopsy). See a l s o F i g u r e 4 1 f o r t o t a l s . Two graph charts have been prepared to a i d i n asses s i n g the c h a r a c t e r i s t i c s of experimental pseudotuber-c u l o s i s i n the group of twenty i n o c u l a t e d canaries. Figure MAIN GROUPING CRITERIA FOR GROUPING SUB GROUPING CRITERIA FOR SUBGROUPING BIRD NO. RECOVERED DIED A 16 birds Shedders. A l l inoculated bir d s . A l l shed detect-able numbers of P. pseudo on each of at least three' days between 1st post inoculation day and the death of the bird, or end point (exp day No. 25) A - l A l l birds died. High 3" faecal counts u n t i l death. 16 4 birds Culture recovered at 17 postmortem. Monophasic. 21 A-2 . No deaths. Faecal counts 1 day 13 stayed high u n t i l 'recovery'4 day 15 8 birds point. ' No exacerbations. 5 day 21 Monophasic. No recovery of 8 day 18 culture on autopsy. 12 day 20 14 day 17 18 day 12 23 day 14 A-3 No deaths. Faecal counts 2 day 21 showed tendency to 7 day 15 4 birds remissions and e x a c e r b a t i o n s l 3 day 18 Diphasic (7 & 13) or 15 day 19 Triphasic.(2 & 1 5 ) . No recovery on autopsy. day 14 day 23 day 23 day 23 B 4 birds C 4 birds Non-shedders. A l l inoculated. None shed detectable P. pseudo on any of 20 days of faecal t e s t i n g 6 11 19-22 Non inoculated Non shedders 10 9 20 24 TABLE X V I GROUPING OF TWENTY-FOUR EXPERIMENTAL BIRDS ACCORDING TO FAECAL DISSEMINATION DATA O A . l A. 2 B COMPUTED FAECAL EXCRETION DETERMINATIONS CHANGES IN CONDITION GROSS PATHOLOGY Exp Daily Total Day of Peak Counts Weight Weight Day of Degree Total Simulative Bird Faecal Positive Peak P. Amplitude Over Loss Gain Death or Other of Count Expont'1 No. Counts Counts Counts (Log) Log 6.0 % °L Sacrifice Spleen Organs Involvement Per Bird 3 F 10 9 12 6.25 1 16.3 14 ++++ Liver + 2.38 x 10 6 16 M Crop +-H- 10 8 19 19 10 8.1 15 21.8 23 +++++ Heart + 5.02 x Larynx Thymus + Caecum ++++ c 17 M 19 6 21 6.75 i 27.6 23 +•*-++ Liver ++ 6.73 x Peritoneum++ Breast muscle tumor 7 21 M 19 17 19 7.3 4 40.6 23 +++ Mandible ? 3.66 x io 7 1 F 20 8 11 5.0 0 21.9 S-25 ++ n i l 2.03 x 4 F 20 9 10 6.3 1 17.6 S.25 +++ n i l 2.37 x 10? 5 F 20 10 10 6.7 2 7.6 S-25 +. n i l 8.19 x 8 F 20 12 11 6.4 4 11.6 S.25 + n i l ' 1.26 x 10^ . 12 F 20 12 15 7.0 3 22.1 S.25 +++ n i l 1.38 x io] 14 F 20 10 8 6.75 1 11.6 S-25 n i l 6.49 x 10fi 18M 20 7 6 5.5 0 11.2 S-25 + n i l 7.9 x 105 23M 20 8 9 7.6 6 7.0 S-25 n i l 9.02 x 10 7 2 F' 20 11 6 6.4 1 > 6.1 S-25 +++ n i l 3.29 x io| 7 F 20 5 10 5.1 0 11.8 S-25 — • n i l 1.74 x 10^ 13F 20 3 10 5.1 0 5.7 S-25 ++++ n i l 1.39 x 10 5 15F 20 4. 6 4.3 0 11.7 S-25 i • n i l 2.2 x 10" 6 F 20 0 0 0 28.6 S-25 n i l n i l 1IF 20 0 _ 0 0 6.6 S-25 __ n i l . n i l 19M 20 0 _ 0 0 3.4 S-25 _ n i l n i l 22F 20 0 - 0 0 ^•5 S-25 • - n i l n i l TABLE XVIllATA SUMMARY SHEET F0R_ TWENTY INOCULATED BIRDS (EXPERIMENT f C ) I O ON Figure 21 Canary No. 3 Record of Faecal Shedding 22 Canary No. 16 Record of Faecal Shedding S t a r t Faecal C o l l e c t i o n ! Day of S a c r i f i c e LOG VIABLE ORGANISMS SHED DAILY IN FAECES CRAMS TOTAL FAECAL DRY MATTER EXCRETED/DAY DEATH DUE TO INOCULUM Inoculation Period ( > .8 .7 .6 .3 •* .3 . 2 jj! 1 i a •°i. . 9 a i .7 ' ' i I I \ .4 j .3 j .2 ' 8 12 16 - EXPERIMENTAL'DAYS Flaure 2^ Canary No. 17 Record of Fa oca 1 Shedding S t a r t Faecal CoLlectlona Day of S a c r i f i c e LOG VIABLE ORGANISMS SHED DAILY IN FAECES CRAMS TOTAL FAECAL DRY MATTER EXCRETED/DAY * DEATH DUB TO INOCULUM A A t \ /' Inoculation P.rlod 8 12 16 EXPERIMENTAL' QAYS 1.7 1.6 .3 1.4 1.3 2 1 1.0 .9 .8 .7 .6 .3 .4 .3 .2 .1 S t . r t FaacaL C o l l e c t i o n * Day of S a c r i f i c e LOG VIABLE ORGANISMS SHED — D M L V " VAECES CRAMS TOTAL FAECAL DRY • " - H A T T E R IXI • DEATH DUB TO INOCULUM . Inoculation P.rlod ( > 1 2 3 . .24 8 12 16 20 EXPERIMENTAL DAYS Canary No.'21 Record of Faecel Shedding 1.7 1.6 1.3 1.4 1.3 1.2 1.1 1.0 .9 .8 .7 .3 ,4 .3 .2 .1 St a r t Faecal C e l l a e t l o n a Day of S a o r l f l e . LOG VIABLE ORCANISMS SHED DAILY IN FAECES CRAMS TOTAL FAECAL DRY MATTER EXCRETED/DAY * DEATH DUE TO INOCULUM Inoculat Parlod < > -2TT EXPERIMENTAL DAY8 Figures 21-24. Grpup A - l . Four deaths due to experimental pseudotuberculosis. A l l shedders. Graph charts of data collected dally on faecal counts of viable P. pseudotuberculosis f and faecal output in terms of dry matter. t l S M I l 25 Canary Ho. L Racord of Ta»aal Shaddlng S t a r t Faacal C o l l a c t l o n a . LOG VIABLE ORCANISHS , SHED DAILY IN FAECES CRAMS TOTAL FAECAL DRY MATTER EXCRETED/DAY Day of S a e r l f i x I1 12 16 EXPERIMENTAL DAYS Trrr L.7 1.6 .3 . 4 1.3 1.2 1.1 1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 Usui. Canary No. 5 R*«ord of r a s c a l Studding Scare Faocal C o l l a c t l o n a Day of S a c r i f i c e LOC VIABLE ORCANISHS SHED DAILY IN FAECES CRAMS TOTAL FAECAL DRY MATTER EXCRSTED/DAY Inocu la t ion Far lod < » 1.6 j ...-! ... | 1.3 ;. «•* t' i . i >. ai • O i 1.0 .9 ""j .8 .7 .6 .3 .4 . } 8 12 16 EXPERIMENTAL DAYS 26 Canary Ho. 4 Racord of Faaca l S h a d i n g s t a r t F a . c . 1 C o l l a c t i o n . Day o( S a c r i f i c e LOO VIABLE ORGANISMS SHED DAILY IN FAECES CRAMS TOTAL FAECAL DRY — MATTER EXCRETED/DAY I n o c u l a t l i f i P . r l o d I 1.7 1.6 .3 i . 4 . i . 0 | • ' H .8 u .7 .6 .5 . 4 .3 . 2 .1 Ilgur. 8 12 16 20 24 25 EXPERIMENTAL DAYS Canary Mo. 8 Racord of Faaca l Shaddlng -N.J Stare Faaca l Co l l aoc lona Day of S a e r l f l o a LOG VIABLE ORCANISHS SHED DAILY IN FAECES -CRAMS TOTAL FAECAL DRY HATTER EXCRETED/DAY Inocula Far lod < ) 1.8 1.7 . 1.6 1.3 . 1.4 . 1.3 . 1.2 i.o 2 •'! .8 ; .7 .6 .3 .4 .3 .2 .1 8 12 16 EXPERIMENTAL DAYS gures 25-22 Group A-2. Four of e i g h t monophasic shedders which recovered. Graph charts of data c o l l e c t e d d a i l y on f a e c a l counts of v i a b l e P. pseudotuberculosis., and f a e c a l output in.Terms of dry matter. 109 3 $ 25 Canary No. 12 Record of faecal Shedding Start Faecal Collection* Day of Sacrifice LOG VIABLE ORGANISMS SHED DAILY IN FAF.CES CRAMS TOTAL FAECAL DRY MATTER EXCRETED/DAY Inoculation Period < > 1.6 1.3 1.4 | 1.2 |~! 2 .8 5 .7 .6 .3 .4 .3 .2 .1 8 12 16 EXPERIMENTAL DAYS ; S Usui. Cenary No. 14 Record of Feecel Shedding Start Faecal Collectlone Day of-Secrlflce LOG VIAPLE ORGANISMS SHEO DAILY IN FAECES GRAMS TOTAL FAECAL DRY MATTER EXCRETED/DAY lnoculatlo Period < > 0 1 2 3 4 1.7 1 1.6 1.5 I 1.4' l 1.3 ! ,|; 1.1 >. • s 1.0 i .8 l i .7 S i | .3 ; . •J i .2. i i .i .> i 12 16 EXPERIMENTAL DAYS 21- Canary No. 10 Record of Faeca l Shedding" Flaure Canary Ho. 23 Record of Faecal Shedding Start Faecal Collection* Dev of S a c r i f i c e LOG VIABLE ORCANISMS SHED DAILY IN FAECES CRAMS TOTAL FAECAL DRY HATTER EXCRETED/DAY Inocu la t ion Per iod < > M . ! 1.7 ! 1.6 : 1.3 | i 1.4 1.3 2 ,..g •»g . 8 ° .7 .6 .5 .4 .3 : •2 I I . i ! 8 12 16 EXPERIMENTAL DAYS Start Faecal Collection. Day of Saorl f le . 100 VIABLE ORGANISMS SHED DAILY IN FAECES GRAMS TOTAL FAECAL DRY MATTER EXCRETED/DAY Inoculacl >n Period < > A 1.6 1.3 1.4 . 1.2 | 1.1 > ! 1.0 j .9 | .8 t .7 .6 .5 .4 .1 .2 .1 « 12 16 EXPERIMENTAL DAYS F i g u r e s 29-32 Group A-2 (contd). Four of eight monophasic shedders which recovered. Graph charts of data c o l l e c t e d d a l l y on f a e c a l caunts of v i a b l e P. pseudotuberculosis, and f a e c a l output i n Terras or dry matter. ' lisaa 23 Canary K o . 2 Racord b( Faaca l Shaddlng Fijtura Canary Ho. 7 Racord of f a a c a l Shaddlng Scare r a s c a l Co l l ee t l one Day of S a c r i f i c e LOG VIABLE ORCANISHS SHED DAILY IN FAECES CRAMS TOTAL FAECAL DRY HATTER EXCRETED/DAY Inocu la t i on Fa r lod < > A 0 1 2 1 4 ~~i 12 I t EXPERIMENTAL DAYS 1.7 1.6 1.3 1.4 i . ; 1.2 B i i . i g i . o 5 c .9 „ .8 *» .7 .6 .5 .4 .3 : i .2 f J . 1 i ' I. . f F i g u r e ^ 5 Canary No. 13 Racord of Faacal Shedding S ta r t Faaca l Co I Wot Ion Day ot S a a r l f i o a LOG VIABLE ORGANISMS , SHED DAILY IN FAECES Inocu la t ion Per iod ( ) b i i i 4 1 T n ' is 1—nr EXPERIMENTAL DAYS 8 I Sta r t Faacel C o l l e c t i o n . Dey o f S a e r l f l e i LOO VIABLE ORGANISMS SHED DAILY IN FAECES CRAMS TOTAL FAECAL DRY , MATTER EXCRETES/DAY Inoculec lon Per iod A 6 12 EXPERUSNTAL DAYS 1.7 1.6 1.3 1.4 1.3 1.2 1.1 1.0 . 9 .8 .7 .6 .3 .4 .3 .2 . 1 Canary No. 15 Racord of Faaca l Shaddlng 1.8 1.7 j 1.6 l . J j 1.4 { 1.3 1.2 i j 1.1 i a 1.0 . 9 t o i 3 i i .8 1 LOG .7 .6 .5 | .4 .3 '< .2 ! .1 » Sta r t Faaca l C o l l a o t l o n Day of S a c t i i i c a LOG VIABLE ORGANISMS SHED DAILY IN FAECES CRAMS TOTAL FAECAL DRY .HATTER EXCRETED/DAY Inocu la t ion Par lod V i \ : 0 1 2 3 4 Ti rr*—Tir EXPERIHEKTAL DAYS T l 23 1.8 1.7 1.6 1.3 1.4 1.3 1.2 1.0 . 9 .8 .7 .6 .3 .4 .3 . 2 .1 F i g u r e s 33-3^ Group A-3. Four d i - and t r i p h a s i c shedders which recovered. Graph charts of data c o l l e c t e d d a i l y on f a e c a l counts of v i a b l e P. pseudotuberculosis, and f a e c a l output In Terms or dry matter." **1 CD CQ I -f=-o LOG VIABLE ORGANISMS •4 H O flO CO O ^ 4 CD £ CD O O CD c+ (-> CQ o to O o • C Hi ») cH f d < C+ ^ c P to o C+ CD £ & o ^ i—1 M> CD h* P< 3 c-t i * T j CO O CD • c+ O CD C s CO co o a CD O c+ o C H (D Ml pj \-> pi O CD Pi c+ O 3 C c+ O << CC CD 3 CD PJ 1 3 4 CO P o p , ? c+ C CO CD trt CD O M PJ on ^ CD • CD O CD 3 • P, CD CD O fD M P 15 a? *-»0» EG >3S O* to <0 O CKAHS FAECM. DRY HATTER LOG VIABLE asauosKS -c -J»-" ~ O ^- „ CBAMS FAECAL DRY MATTER LOG VIABLE ORGANISMS o 4! 05. S 2 5 Figure kl Excretion of viable Pasteurelia pseudotuberculosis "by inoculated birds totals for twenty experimental days (exponential values) 113 ^2 i l l u s t r a t e s the d a i l y t o t a l s of shedder b i r d s ( s o l i d l i n e ) (Group A Table XVI) out of the t o t a l s u r v i v o r s of the group of s i x t e e n b i r d s which shed the t e s t organisms at some stage d u r i n g the experiment ( i n t e r r u p t e d l i n e ) . I t w i l l be noted that the peak of e x c r e t i o n of the organisms i n terms of num- bers of b i r d s shedding was reached on the te n t h day. Since the mean p o i n t of peak e x c r e t i o n i n terms of numbers of  organisms f o r the s i x t e e n shedder b i r d s i s 10.2 days i n t o the experiment (the median and mode being 13*5 a n < i 10.0 .-res-p e c t i v e l y ) i t seems l i k e l y that t h i s f i g u r e (10 days) bears r e l a t i o n s h i p to the maximum b a c t e r i a l i n v a s i o n of the b i r d s as a group and hence of the greatest bacteremia. This i n tu r n suggests that the average i n c u b a t i o n p e r i o d of the exp-erimental disease by the o r a l route c o u l d best be s t a t e d as 10 days. For t h i s purpose i n c u b a t i o n p e r i o d i s defined as the time I n t e r v a l between f i r s t i n o c u l a t i o n and the appearance of maximum f a e c a l e x c r e t i o n of the t e s t organism. In F i g u r e 4-3 the t o t a l d a i l y number of shedder b i r d s i s again p l o t t e d ( s o l i d black l i n e ) , . This l i n e descends f i n -a l l y to zero at the 2^th experimental day as a r e s u l t of four deaths ( d o t t e d l i n e ) and twelve " r e c o v e r i e s " ( i . e . b i r d s which have ceased to shed). The s o l i d red l i n e p l o t s the d a i l y i ncrease i n numbers of r e c o v e r i e s (up to a t o t a l of twelve). These two s o l i d l i n e s cross on the 18>th experimental day which l t i s suggested might be considered as the p o i n t of maximum e f f e c t i v e immunity i n the group of a f f e c t e d ( f e c a l 11* NUMBERS OP BIRDS ACTUALLY SHEDDING EACH DAY TOTAL 'NUMBERS SURVIVING CARRIER OR SHEDDER BIRDS Day of Sacrifice Total Numbers Inoculated Orally (Exp days 1 - 3 ) one j death three "| deaths | 1 2 8 12 EXPERIMENTAL DAYS 2 I T 5 1 Four of the total of twenty-four birds were non-inoculated controls 2 Four of the total of twenty-four birds were inoculated non-shedders Figure k2 D a i l y record showing numbers of b i r d s shedding (Group A-Table XVI) i l l u s t r a t e d by a graph chart 115 2k 23 22 21 20 19 18 17 16 15 Day of Sacrifice> TOTAL SHEDDER BIRDS LESS RECOVERIES TOTAL NOS. RECOVERED BIRDS (CEASED TO SHED) TOTAL NUMBERS SURVIVING SHEDDER BIRDS Total Numbers Inoculated Orally (Exp days 1 - 3 ) one lj.ea.t_h  three deaths Pinal Paecal Collection i 2 3 h 5 6 7 8 9 lb l i 13 lh 15 lo 17 1319 2021 22 23 2^25 EXPERIMENTAL DAYS 1 Pour of the total of twenty-four birds were non-inoculated controls 2 Pour of the total of twenty-four birds were inoculated non-shedders Figure kj D a i l y record showing numbers of b i r d s r e a c h i n g the p o i n t of "recovery" i n terms of c e a s i n g to shed v i a b l e P. os e u d o t u b e r c u l o s i s (Group A - Table XVI) 116 shedder) b i r d s . The median p o i n t between when the f i r s t and the l a s t b i r d s reached the recovery p o i n t was s i x t e e n days. I t can r e a d i l y be seen that i f canaries contact P. pseudo-t u b e r c u l o s i s , become i n f e c t e d , shed v i a b l e organisms, and do not recover on the average u n t i l s i x t e e n to eighteen days l a t e r , the chances of the i n f e c t i o n spreading to other b i r d s i s p o t e n t i a l l y high. (e) Gross pathology and c u l t u r a l recovery (Figures 44 - 48) The f o u r b i r d s i n Group A - l , a l l of which d i e d , were found to show t y p i c a l pseudotuberculosis nodules i n v a r i o u s l o c a t i o n s , and a pure c u l t u r e of P. pseudotuberculosis was checked b i o c h e m i c a l l y and found s t i l l capable of fermenting maltose. Of the remaining s i x t e e n i n o c u l a t e d b i r d s , twelve were shedders, and f i v e of these showed d e f i n i t e gross s p l e n i c l e s i o n s , while s e v e r a l more e x h i b i t e d a s l i g h t degree of s p l e n i c enlargement and/or congestion. None of the four non-shedders showed any such changes on autopsy. These f i n d i n g s are r e c o r -ded i n Table XVII. P a r t i c u l a r a t t e n t i o n i s d i r e c t e d to B i r d No. 16 which showed l e s i o n s i n the caecal lumena ( F i g u r e 4 7 ) . ( f ) Histopathology Pseudotubercles were studied i n the caecum of one b i r d and i n the spleens of a l l which died. The l e f t c aecal lumen of canary No. 16 ( F i g u r e 49) was noted to be f i l l e d w i t h Figure 44 Experiment C. Bird No. 16. Autopsy showing trmendous size of affected spleen. Figure * 5 Experiment 0 . Bird No. 1 6 . Close up of spleen which is made up of a solid mass of expanding pseudotubercles. Figure 4-5. Figure 46 Experiment C. Bird No. 16. Lesion in thymus area of neck. Figure 4-7 Experiment C. Bird No. 16. Close up view of swollen and abscessed cecal buds. The l e f t cecum can be seen to have a distinct yellow nodular abscess at the blind extremity. This shows through the serosa. This bird excreted dally counts of Pasteurella pseudotuberculosis of up to Log 5.1 for nineteen consecutive days until i t s death. Figure k6 Figure 4s" Experiment C. Bird No. 17. The autopsy disclosed lesions in the spleen and breast muscles. This type of spleen with yellow to white pseudotubercles,is pathognomonic for pseudotuber-culosis in the canary. Ifl spite of these lesions, this bird did not oommence faecal shedding of the study organism until twelve days following conclusion of the course of three peroral Inoculations. It then came to a daily peak of Log. 6.75 P. pseudotuberculosis organisms shed 48" hours before death. I see Figure 23). vFigure 4_. 120 a mass of macrophages which had i n f i l t r a t e d and rep l a c e d the mucosal t i s s u e s and extended hack i n t o the muscularis mucosa ( F i g u r e 5°) • C e n t r a l l y l o c a t e d was a n e c r o t i c centre-which ' :s t a i n e d pink w i t h H. and E. (F i g u r e 50). At the periphery was a r i n g of l a r g e macrophages showing marked g r a n u l a r i t y i n d i c a t i v e of ba c t e r i o p h a g i a ( F i g u r e 51)- Many g i a n t c e l l s of the Langhans type (F i g u r e 52) were noted i n the median area. The spleen showing g r e a t e s t enlargement was from the same b i r d ( F i g u r e ^5). The microscopic p i c t u r e was t y p i c a l of other spleens. The changed organ was seen to be made up of a mass of expanding granulomas w i t h oaseating centres (Flg - r ure 53) • An intense mass of macrophages wi t h l e s s e n i n g deg-rees of degeneration extended out In c o n c e n t r i c r i n g s from the n e c r o t i c centre. At the periphery a r i n g of such c e l l s contained ingested b a c t e r i a ( F i g u r e 5 4 ) . The same t i s s u e s when s t a i n e d w i t h Twort's method using f a s t green f o r a coun-t e r s t a i n , showed the c e n t r a l n e c r o t i c zone s t a i n i n g blue and the p e r i p h e r a l g i a n t c e l l s w i t h t h e i r b a c t e r i a l l o a d showing as an intense red r i n g on low power (Fi g u r e 5 5 ) . High m a g n i f i c a t i o n allowed i l l u s t r a t i o n of the mass of r e d - s t a i n -i n g b a c t e r i a showing the t y p i c a l p a t t e r n of b i p o l a r l t y ( F i g u r e 5 6 ) . H 8c E *0X Figure * 9 Experiment C. Canary No. 16. Exp-erimental pseudotuberculosis. The two photomicrographs on th i s page i l l u s t r a t e the histopathology of the caecal abscess. Low power cross section of colon and caecal lumena shot*s the pink s ta in ing necrot ic centre of the tubercle abscess i n the r igh t caecum. H & E 15QX Figure 50 Experiment C. Canary No.•16. A close up photomicrograph.of the abscess i l l u s t r a t e d i n Figure *9» The necrot ic centre', i s at the r i g h t , a mass of mac-rophages ( including giant c e l l s ) , i n mid zone, and part of the r i n g of giant c e l l s with phagocytosed bac te r ia (note blue smudging) at l e f t . Figure 50. H & E X 1500 Figure 51 Experiment 0. Experimental pseudotuberculosis canary N0.16. High power photomicrograph of the caecal abscess shows a part of the giant c e l l ring which apears in Figures 49 and 5°. Numerous blue bi-polar-stalning v a c i l l i can be noted engulfed by the giant c e l l s . H & E X 1500 Figure 52 Experiment. C. Experimental pseudotuberculosis canary No.l6. High power photomicrograph of mass of giant cells in the mid-zone of the cecal abscess illustrated in Figure 50. Figure 51 Figure 52 H & E X 30 Figure 53" Experiment C. Experimental pseudotuberculosis canary No.16. Low power photomicrograph of section of spleen which was illustrated in gross in Figure kk. The concentric rings Indicate the expanding development of the pseudotubercles with their necrotic centers, and peripheral rings of giant c e l l macrophages containing large numbers of b a c i l l i . H & E X 150 Figure 5^  Experiment 0. Experimental pseudotuberculosis canary No. 16. This high power photomicrograph shows the various zones of the pseudotubercles which are i l l u s t -rated in Figure 53• The necrotic centre is at the l e f t and the giant c e l l ring at right. Figure 54 Twort«e X 50 Figure 55 Experiment C. Photomicrographs of the spleen illustrated in Figures 53 and 5*. The stain used for this section is O l l e t t 1 s modification of Twort's method. The necrotic centre retains the crystal violet, the gram negative phagocytosed "bacteria are stained red, and the cytoplasmic areas take the Fast green counter stain. Twortrs X 1200 Figure 56 Experiment 0. Photomicrograph showing high pwer view of a section of the spleen in Figure 55 (above). The neutral red staining bacteria are packed into . the macrophages. The staining bi-polarity is clearly visible in this picture. This canary (N0 .16) was the heaviest faecal shedder of Pasteurella pseudo-tuberculosis. Figure 55 Figure 56" 125 Summary and Conclusions (a) On the c l i n i c a l appearance during the experiment I t was concluded t h a t the s u p e r f i c i a l appearance of the b i r d s was not a r e l i a b l e i n d i c a t i o n of the. outcome of the experiment. Many b i r d s which showed h i g h f a e c a l counts f o r the Inoculated organism and one of the b i r d s which di e d (No. 1 6 ) , showed no t a n g i b l e c l i n i c a l s i g n s . Both i n o c u l a t e d and c o n t r o l b i r d s seemed to have t h e i r bad days. The spread of a n a t u r a l l y o c c u r r i n g c o n d i t i o n of s o f t s t o o l s among eleven of the twenty-four b i r d s , I n c l u d i n g three of the c o n t r o l b i r d s , i s mentioned although i t appears not to have i n f l u e n c e d the course of the experimental disease. (b) On the course of the disease as measured by f a e c a l data and weight In summary i t i s f e l t that the experimental disease can be st u d i e d reasonably w e l l i n a small species (which oannot be handled without a l t e r i n g the disease course) by t a k i n g d a i l y twenty-four-hour f a e c a l samples. The r e s u l t s of f a e c a l colony counts f o r v i a b l e P. pseudotuberculosis organisms suggest the f o l l o w i n g conclusions: —• The inc u b a t i o n p e r i o d i s probably about ten days, i n an experimental i n f e c t i o n of t h i s s e v e r i t y ( 2 0 per cent 126 mortality, with 50 per cent of the survivors reaching recovery point by 2§ weeks). The height of effective immune activity is possibly reached in about Z\ weeks from the f i r s t day of inoculation of l i v e culture by the oral route. The experimentally infected canary disseminates large numbers of viable P. pseudotubercu!os1s bacteria for extended periods even i f i t later recovers from the infection. There was no evidence, in this experiment that the canary becomes a long-term infected carrier. However, the course of the disease to death in birds which are heavy faecal disseminators can extend to over three weeks. This may be of great epizootlological import in the canary species, and could have zoonotic importance, as recently suggested by the findings.of Paterson and Gook (1962) which are referred to in Appendix Section on pseudotuberculosis in man. Tubercles or abscesses containing proliferating P. pseudotuberculosis were found in.the mucous membrane of the bowel (specifically the caecum, a vestigial organ). This condition was seen in three birds with the naturally occurring disease from aviaries No. 1. and No. 3 . The caecal abscess was postulated at that time to be Indicative of a b i l i t y to excrete extremely high counts of the causative organism, in 127 the faeces. This l e s i o n was produced i n the experimental disease i n one b i r d , and l o g a r i t h m i c f a e c a l counts of over 8\0 were recorded i n as l i t t l e as 0.66 gm (DM equivalent) of faeces excreted over a twenty-four hour p e r i o d . The experimentally i n f e c t e d canary continues to eat during the course of the disease, which i n turn supports i t s a b i l i t y to disseminate s i g n i f i c a n t numbers of v i a b l e org-anisms from the bowel. -~ The experimental i n f e c t i o n of canaries by the o r a l route u s i n g a homologous Type I s t r a i n ( i s o l a t e d i n B r i t i s h Columbia) apparently r e q u i r e s a heavy dosage to achieve a uniform e f f e c t . The dose l e v e l s here used, w i t h extension over a three-day p e r i o d , but w i t h no i n t r o d u c t i o n of any g a s t r o - i n t e s t i n a l i r r i t a n t , caused SO per cent of twenty b i r d s to become i n f e c t e d . I t i s suggested that i n the n a t u r a l disease w i t h i t s f r e q u e n t l y h i g h m o r t a l i t y (as occurred i n B r i t i s h Columbia i n 1958) the pathogenesis i s based i n p a r t on p r e d i s p o s i n g s t r e s s f a c t o r s . (c) On the pathology of experimental pseudotuberculosis i n canaries In t h i s experiment, p a t h o l o g i c a l l e s i o n s were noted i n f o u r b i r d s that d i e d and i n f i v e others which were s a c r i -f i c e d . There appears to be no s i g n i f i c a n t d i f f e r e n c e between 128 the experimental disease and the natural disease, in that the most concentrated focus of change is the spleen. Of probable great importance was the finding of abscess tubercles in the caecum of one of the birds (which was the heaviest faecal disseminator of the causative organism). This type of pathology has also been noted in three canaries from different avaries suffering natural epizootics (See Tables VTI and XVII). 129 DISCUSSION AND SUMMARY OF STUDIES ON EPIZOOTIOLOGY AND EXPERIMENTAL INFECTIONS OF PASTEURELLA PSEUDO-TUBERCULOSIS 1. Pseudotuberculosis is now the universally recognized term for infections with Pasteurella pseudotuberculosis. This term should be reserved s t r i c t l y for such infections (Meyer 1959) . 2 . The spectrum of hosts for this pathogen is perhaps the widest encountered for any bacterium (Burrows 1963) and the known hosts include man, in particular children, in whom appears a non-fatal mesenteric adenitis which can c l i n i -cally resemble acute appendicitis. 3 . For the last eighty years, since its discovery, P. pseudotuberculosis has been associated primarily with the continent of Europe. Of relative interest also is the apparent absence of detectable pseudotuberculosis in the Far East, as well as in South Africa, Madagascar, and Cen-t r a l Africa (Devignat 1953) . k. Three canary epizootics of pseudotuberculosis occurred in the Vancouver and Vancouver Island areas in 1958. There was every appearance that the foc i of the infections were endemic to this area. The presence of f i e l d mice in the aviary (not actually identified but in one case sick or 130 or dying) appears to have been closely associated in two ^ oases, and could have been a factor in the third case (Aviary No. 2). Although data relating to a possible con-current f i e l d mouse epizootic is not available for any of the immediate areas concerned, there i s one recorded obser-vation for the Agassiz area of the Praser Valley, a point about 80 miles from Vancouver. A trained agricultural observer (Clarke 1961) supplied evidence to suggest that the population of short tailed f i e l d mice in that area reached a peak in the f a l l and winter of 1957-58. Swampy pasture ploughed that f a l l was found to have the turf riddled with mouse nests. Several, sleek almost tame coyotes followed the plough picking up mice from the furrow. Farmers complained of mouse damage to new stands of grass and clover. In the f a l l of 1958 no mice were seen. The coyotes disappeared and have not been seen down in the val-ley since. 5« From this one might conjecture that a major epizootic of transmissible disease (pseudotuberculosis?) probably occurred in one or more species of rodents in the lower mainland area. This probably involved f i e l d mice in the vici n i t y of the epizootics prior to or at the time of the f i r s t losses in canaries. This was assumed to be the case at the time of the f i r s t epizootic but in spite of requests no f i e l d mice were procurable for examination and time did 131 not allow execution of the survey that was obviously des-irable. 6. Examination of the literature made i t appear that pseudotuberculosis infections were rare in. birds, and in mammals in North America, other than in guinea pigs and to a lesser extent chinchilla colonies. The recent associa-tion of guinea pig infections with the feeding of f i e l d greens soiled by pigeon faeces (Paterson and Cook 1962) in England, and the recent grackle epizootic in Maryland (Clark and Locke 1962) when combined with the British Columbia data, could suggest that avian and rodent epizootics may be occurring more frequently, or have been overlooked in North America. In.Table XVIII which accompanies this section, I have assembled, the names of some of the known hosts for P. pseudotuberculosis. These hosts are arranged so as to f a l l into one or more of four different categories thus:-I. Suspected reservoir (carrier) hosts II . Epizootic species in rural (feral) and urban areas III. Epizootic domesticated or farm species IV. Sporadic infection species (probably often unnoticed) The principal hosts are underlined and the groups are inter-connected with directional arrows to il l u s t r a t e what I feel 132 A S U S P E C T E D R E S E R V O I R ( C A R R I E R ) H O S T S W I L D R O D E N T S W I L D B I R D S O T H E R S F I E L D M I C E Rats? Others? PIGEONS (England) Others Owls (Sweden) Crows > 4—^-H A R E S (Europe) Others? f I I EPIZOOTICS IN RURAL AND URBAN AREAS FIELD MICE Beaver Muskrat? Others PIGEONS & DOVES GALLIFORMES Pheasant P a r t r i d g e PASSERINES Others? -> HARES (Europe) Rabbits? Others? » \ \ « r ' r I I I EPIZOOTICS IN DOMESTICATED (OR FARM) SPECIES GUINEA PIGS (worldwide) CHINCHILLAS (N.America) Microtus [agreBtls;| ("Oxford, Eng) C h i t t y 1962 . CANARIES MINK (Denmark) ©THER\ CAPTIVE HARDBILLS TURKEYS CHICKEN'S OTHERS A IV SPORADIC INFECTIONS (OFTEN UNNOTICED) \ CATTLE SHEEP CATS \ \ 4 MAN (Germany, France England, U.S.A. Canada, others) fogs' PRIMATES 7 4 TABLE XVIII CONJECTURAL PATHWAYS OF TRANSMISSION OF PASTEURELLA PSEUDOTUBERCULOSIS ASSOCIATED WITH EPIZOOTIC AND SPORADIC INFECTION 133 to be the p r i n c i p a l pathways of transmission of pseudo-tuberculosis i n f e c t i o n . One s o l i d diagonal l i n e i l l u s t r a t e s the probable spread of in fec t ion from f i e l d mice to canaries i n outside or access ib le av ia r ies which could have taken place i n B r i t i s h Columbia. An interrupted l i n e shows the conjectural r e l a t i onsh ip between canaries and man (because of c lose s p a t i a l contact) which i n part led to the decis ion to carry out experiments wi th a r t i f i c i a l l y induced in fec t ion i n canar ies . ?• The canary appears to be highly susceptible to pseudo-tuberculosis Judging by the ear ly European l i t e r a t u r e and the experiences i n Vancouver. Some predisposing stress factors appear to be necessary for ah-epizootic occurrence; but mor t a l i t i e s are extremely high although the disease pursues an ins id ious course of up to several months wi th in the av i a ry . 8. A prel iminary study of the three epizoot ics l ed into experimental work with canaries transferred from epizoot ic premises to the laboratory for c loser study. Prom the point of view of l a t e r a l spread of in fec t ion among canaries, and from the considerations of human contact a phase was en-tered where the studies were based on experimental i n f e c t -ions . 1 3 * 9 . I n d i c a t i o n s were t h a t the spread of i n f e c t i o n to sus-c e p t i b l e hosts (when s u i t a b l y s t res sed) must take p l a c e through i n g e s t i o n of f a e c a l m a t e r i a l . (Meyer 1958 ) (Man-n i n g e r and Mocsy 1 9 5 6 ) . The gross p a t h o l o g i c a l l e s i o n s d i d not appear t o f i t a p a t t e r n of h i g h f a e c a l shedding u n t i l i t was n o t i c e d t h a t some b i r d s (about 5 per cent o f those examined) showed the presence of c a e c a l abscesses , l e s i o n s w i t h an i n t e r e s t i n g p a r a l l e l to a l i m e n t a r y and as soc-i a t e d a d e n i t i c l e s i o n s i n the human. I n a c a r e f u l l y monitored experiment , the r a t e and p a t t e r n of f a e c a l shedding o f v i a b l e P . p seudotubercu los i s was recorded over a t h r e e week p e r i o d , f o l l o w i n g a t h r e e day o r a l i n o c u l a t i o n course i n twenty b i r d s . There were f o u r u n i n o c u l a t e d c o n t r o l b i r d s i n the t e s t e d group. 1 0 . Gross p a t h o l o g i c a l and h i s t o p a t h o l o g i c a l changes were assessed and eva lua ted i n terms of the c h a r a c t e r i s t i c s o f f a e c a l shedding and subsequent death or recovery ( b i r d s which ceased t o shed and harboured no v i a b l e organisms i n t h e i r t i s s u e s a t the 2 5 t h exper imenta l d a y ) . 1 1 . The s t r i k i n g s i m i l a r i t y o f gross l e s i o n s between spon-taneous and exper imenta l p seudotubercu los i s gave the impress ion t h a t the o r a l route i s indeed the n a t u r a l one i n c a n a r i e s . T h i s was f u r t h e r supported by the s c a l e of f a e c a l shedding of P . p seudotubercu lo s i s from the f o u r f a t a l l y a f f e c t e d b i r d s (20 per cent) and the twelve t r a n s i e n t l y a f f e c t e d b i r d s (60 per cent) which t o t a l l e d s i x t e e n ( 8 0 ; p e r cent) of those 135 inoculated. The incubation period appeared to be about ten days with the mean recovery point for non-fatal cases being about one week l a t e r . Faecal shedding started early and i n most cases stayed at high l e v e l s u n t i l death or recovery occurred. (Figures 21 - 3 2 ) . Four of the recovered birds showed a ten-dency to regressions and exacerbations of f a e c a l shedding how-ever which might have been re l a t a b l e to the c l i n i c a l course of the disease. (Figures 33 - 3 6 ) . 1 2 . The development ofc c h a r a c t e r i s t i c caeca! lesions i n one of the experimentally infected birds (No. 16) was accompanied by the highest rate of faecal shedding f o r an Individual b i r d (Figures 22 and * 1 ) . I t i s f e l t that probably other grossly undetected lesions occurred i n the caecal ton-s i l s or bowel mucosa of a d d i t i o n a l birds which shed the test organism. Generally speaking the f a t a l cases shed most orga-nisms followed by the steady shedding, then the d i - or t r i -phasic shedders. This trend shows c l e a r l y i n Figure * 1 . 1 3 . In a spontaneous epizootic an incubation period of ten days or more accompanied by high l e v e l s of f a e c a l excretion of f_. pseudotuberculosis. could result i n the more resistant birds being exposed by the o r a l route f o r long periods u n t i l t h e i r resistance broke down. In such a s i t u a t i o n the high mortality rates would be expected since i n a l l p r o b a b i l i t y birds do not 136 begin to develop s p e c i f i c immune mechanisms u n t i l the f i r s t focus of i n f e c t i o n has been e s t a b l i s h e d i n the alimentary t r a c t . 1 This long term exposure was l a c k i n g under the condi-t i o n s of the experimental i n f e c t i o n (Experiment C) which could account f o r the m o r t a l i t y being only 20 per cent. Ik, D i s p e r s a l of b i r d s from an i n f e c t e d a v i a r y could have great p o t e n t i a l danger t o other a v i a r y stocks and humans, i n l i g h t of the prodigious a b i l i t y of the canary t o shed v i a b l e P. pseudotuberculosis organisms i n the f a e c a l m a t e r i a l . This a p p l i e s e s p e c i a l l y i n the presence of caecal abscesses, which were shown to occur i n both spontaneous and experimental can-ary i n f e c t i o n s . 1 5 . Devignat through h i s s t u d i e s of the development and spread of the v a r i e t i e s of the plague b a c i l l u s , P a s t e u r e l l a ( Y e r s i n i a ) p e s t l s . p a r t i c u l a r l y i n the European area; and Burrows and Bacon ( i 9 6 0 ) through comprehensive comparative s t u d i e s which included that of V and W antigens i n the plague b a c i l l u s and i n P. pseudotuberculosis f have expounded and supported the theory that the l a t t e r may be a transmutant form of £. p e s t l s to which i t r e t a i n s an a b i l i t y to r e v e r t . I f t h i s i s so, then two major mysteries may be explained, namely, 1 I n Experiment C, l t was noted that towards the end of the c u l t u r e runs f a e c a l i s o l a t e s were i n a rough form on primary I s o l a t i o n . This could have been due t o the e f f e c t of s p e c i f i c Immune mechanisms. These rough forms were not a c t u a l l y t e s t e d f o r t h e i r expected l o s s of v i r u l e n c e . 137 the 'sudden disappearance of mediaeval plague from Europe 1 (Devignat); and the puzzling alternation: of plague epidemic and quiescence throughout history (Burrows and Bacon). The former occurrence could have arisen from an extension of the Pasteurella (Yersinia) pseudotuberculosis i n f e c t i o n i n rodents i n Europe (aided by avian, and domestic animal e p i -z o o t i c s ) , giving cross-immunity and rendering the t e r r a i n unfavourable f o r the maintenance of plague. These theories appear to be supported by the claims of Russian authors to have achieved i n v i t r o the mutation of P. p e s t i s to the organism of pseudotuberculosis. 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Concerning e p i z o o t i c s of canary pseudotuberculosis. 1 4 5 S t o v e l l , P.L. 1 9 6 3 . Unpublished data. Biochemical f i n d i n g s r e l a t e d to studies reported i n t h i s t h e s i s . S t o v e l l , P.L. and R.J. Avery, i 9 6 0 . Unpublished data. Con-cerning pseudotuberculosis i n chickens i n A l b e r t a . S t o v e l l , P.L. and P.A. Simon, i 9 6 0 . Unpublished data. Con-cerning canary pox i n imported b i r d s . T h a i , E.. 1 9 5 4 . Untersuchungen uber P a s t e u r e l l a pseudotuber- c u l o s i s . Lund. Tha i , E. 1 9 6 1 . Transactions of 1 3 t h Scandinavian Congress of Pathology and Microbiology held at Turku, F i n l a n d . T o t i r e - I p p o l i t i , P. 1 9 2 6 . A n a l l d 'lgiene, no. 6., p. 3 5 9 . T o t l r e - I p p o l i t l , P. 1 9 4 2 . Recent "researches on pseudotuber-c u l o s i s organisms. S e r o l o g i c a l behaviour and s u r v i v a l time. Nuova Vet. 2 0 , 1 3 2 - 1 3 5 . Truche, C. 1 9 3 5 . Pseudotuberculose du cygne. B u l l . Acad, vet. F r . 8 , 2 7 8 . Truche, C. and J . Bauche. 1 9 2 9 . La pseudotuberculose du dindon. Ann. de I ' I n s t . Pasteur, x l l i l . 9 , pp. 1 0 8 1 - 1 0 9 2 . T r u c h e , C and J . Bauche. 1 9 3 3 . Le b a c i l l e pseudotuberculeux chez l a poule et f a l s a n . B u l l . Acad. vet. F r . .6, , 4 3 . Truche, G. et I . Isnar d . 1 9 3 7 . Un nouveau cas. de pseudo-tuberculose chez l a poule. B u l l . Acad. Vet. F r . 1 0 , 3 8 . Truche, M. 1 9 3 8 . La pseudotuberculose chez l e s anlmaux. Rev. Path. comp. 3 8 . 8 7 4 . Urbain, A. et J . Nouvel. 1 9 3 7 . Epidemie de pseudotuberculose chez des toucans de c u v i e r et des toucans a r i e l . B u l l . Acad. vet. F r . 1 0 , 1 8 8 . Van Dorssen, C.A. 1 9 5 1 . P a s t e u r e l l a pseudotuberculosis i n - , f e c t i o n i n pigeons. T i d j s c h r Tlergeneesk. 76. 249-254. Verge, J . et a l . 1 9 3 7 . Un cas.de pseudotuberculose chez l e chat. B u l l Acad. vet. F r . 1 0 , 4 l . Wasielewski and W. Hoffman. 1 9 0 3 . Uber eine seuchenhafte Erkrankung b e i Singvogeln. Arch. f. Hyg., x l v l i . 4 4 - 5 6 . 1*6 Watson, W.A. I 9 6 2 . Ovine A b o r t i o n . The V e t e r i n a r y Record V o l . Z*, No. * 9 . Weidenmuller, H. 1959. P a s t e u r e l l a ps eudotuberculos i s i n f e c t i o n i n animals and man. T l e r a r z t l . Umsoh. 1*, 256-259. Wilson, G.S. and J.A. M i l e s . 1955. Topley and Wilsons P r i n c i p l e s of B a c t e r i o l o g y and Immunity * t h ed. London. Winkle, S. 1955. M i k r o b i o l o g i s c h e und Serologische Diagnostlk mit Berucksichtigung der Pathogenese und Epidemiologic.-Gustav F i s c h e r -Verlag, S t u t t g a r t . Woronoff, A. and A. S i n e f f . 1897. Zur pathologischer Anatomie und B a k t e r i o l o g i e der b a c i l l a r e n Pseudotuberculose. Z e n t r a l b l . f. a l l g . Path. u. path. Anat. 8 : 6 2 2 . Wozniak, D. and J . Chwalibpg. 1958. P a s t e u r e l l a pseudotuber- c u l o s i s i n f e c t i o n i n n u t r i a . Med. vet. Varsovie 16, 11-18. Wramby, G. and M. F r e d r i c s o n . 19*1.- E t t f a l l av pseudotuber-culos hos s v i n . Skand. V e t . t i d s k r . __L, 590. Z e i s s , H. 191*. tfber e>inlge b e i Tierkrankheiten gefundene Erreger aus der Gruppe der hamorrhagischen Septikamle und der Koligruppe. Arch. f. Hyg. 8 * : 1 . Z i n s s e r , H. 1957. " B a c t e r i o l o g y " 11th Edn. p. * 6 6 . Zftrn, F.A. 188*. B l a t t e r f. Geflttgelzucht. Dresden, ..p. 326. Zwlck, W.. 1908. Untersuchungen uber eine Kanarienvftgelseuche. Z e i t s c h r . f. Infekt.-Krankh. d. Haust. *:33.. 14? APPENDIX M a t e r i a l Referred from Page L i t e r a t u r e review on pseudotuberculosis i n mammals and man. (See end of t h i s s e c t i o n ) 5 P r e p a r a t i o n and Use of Wayson's Plague S t a i n : 5 ° D i s s o l v e 0 . 2 gm. f u c h s i n and 0 . 7 5 gm. methylene blue i n 2 0 , 0 ml. of a b s o l u t e - a l c o h o l . Add t h i s dye s o l u t i o n to 2 0 0 . 0 ml. of 5% aqueous phenol s o l u t i o n . F i l t e r . S t a i n smears f o r a few seconds; wash; b l o t dry. F i x a t i o n i s u s u a l l y c a r r i e d out w i t h methyl hydrate. B i p o l a r s t a i n i n g i s enhanced i n P a s t e u r e l l a group organisms. Cook's A c i d Fast S t a i n i n g Method: 50 A t h i n smear made by crushing a small amount of pus between two s l i d e s and drawing them apa r t , or an impression smear, i s f i x e d by gentle heat. The s l i d e i s flooded w i t h a f r e s h l y prepared 1 i n 1 0 d i l u t i o n i n tap water of 1 per cent c a r b o l f u c h s i n . This i s allowed t o s t a i n without heat f o r 3 0 minutes, a f t e r which the s l i d e i s w e l l washed with tap water. D e c o l o r l z a t i o n i s then e f f e c t e d by f l u s h i n g the s l i d e w i t h 0 . 5 per cent a c e t i c a c i d f o r 3 0 seconds, although a longer p e r i o d i s necessary f o r smears of more than o n e - c e l l t h i c k n e s s . The a c i d i s removed by washing and the f i l m i s counterstained f o r 1 0 - 1 5 seconds w i t h L o e f f l e r ' s methylene b l u e . Examination of the f i l m r e v e als that the cytoplasm of the c e l l s and the amorphous debris of the pus are sta i n e d blue or p u r p l i s h b l u e . The c e l l n u c l e i and Past, pseudotuberculosIs are red, the c h a r a c t e r i s t i c b i p o l a r s t a i n i n g of the organisms being w e l l marked. I n t i s s u e smears s t a i n e d by t h i s method from the l i v e r and spleen of mice i n o c u l a t e d w i t h a. s t r a i n of Past, p e s t l s I s o l a t e d from a human source i n N, A f r i c a , i t was of i n t e r e s t that the organisms c l e a r l y ex-h i b i t e d a s i m i l a r degree of a c i d - f a s t n e s s . 148 Referred from Page Table X X I I I Carbohydrate Fermentations as reported by v a r i o u s authors f o r P a s t e u r e l l a pseudotuberculosis. ' ,, (see' over) 51 Table XXIV Other biochemical f i n d i n g s as reported by various authors f o r P a s t e u r e l l a pseudotuberculosis. (see! over) 51 Formula of canary seed mixture used f o r canaries during conduction of Experiment C. 6 l P l a i n canary 70^ Rape 10% Niger 10% F l a x 10% C o n s t r u c t i o n d e t a i l s and environment f o r canary b a t t e r i e s used f o r housing of b i r d s during Experiment C. .... ,. $9 C o n s t r u c t i o n - Two double decked plywood b a t t e r i e s , each w i t h twelve c u b i c l e s of 8" x 12" x 12" dimensions. Hinged doors are i n the back, the f r o n t s are of welded 1" x galvanized,wire c l o t h w i t h expanded apertures f o r access to feed. Water and g r i t are supplied i n g r a v i t y feeders and seed and c u t t l e i n . p l a s t i c cups, a l l mounted on the outside of the w i r e . Droppings c o l l e c t on galvanized s t e e l s l i d e s , which are pro-t e c t e d by removable g r i d s of _§" x §" wire c l o t h ; these s l i d e i n under the wire i n the f r o n t . Environment - The b a t t e r i e s were mounted on a wide s h e l f attached t o the end w a l l of a 9 x 16' room i n a concrete block and pumice walled b u i l d i n g . Room tem-perature is. c o n t r o l l e d by a White Rodgers thermostat attached to two 750 watt Berke glass r a d i a n t heaters c e i l i n g mounted to hang 8 f e e t from the f l o o r and g i v i n g a d i u r n a l v a r i a t i o n of 4 - 8°F. V e n t i l a t i o n i s achieved by a small discharge fan mounted i n the g l a s s b r i c k window above the b a t t e r i e s and a louvred c o l d vent low down oh one s i d e of the room. 1 * 9 Pseudotuberculosis In W i l d Mammals ( i n c l u d i n g f u r bearers chiefly-bred i n c a p t i v i t y ) (a) Fur Bearing Animals An incomplete c o m p i l a t i o n has been made of some r e p o r t s which are a v a i l a b l e on pseudotuberculosis i n t h i s group of animals. The number of r e p o r t s I n v o l v i n g C h i n c h i l l a s , along w i t h observations on pathology and the tendency to recurrence on a farm, a l l suggest that t h i s species i s one of the most s u s c e p t i b l e of animals. (TABLE XIX). G-orham (1955) obtained from Thai ( 1 5 t h I n t . Vet. Congress) Group IV hare i s o l a t e , a s t r a i n of P a s t e u r e l l a pseudo- t u b e r c u l o s i s (No. 32) which the l a t t e r had used f o r a c t i v e ( l i v e ) immunization of guinea p i g s . He was a b l e tonconfirm the i n -a b i l i t y of t h i s s t r a i n of the organism to cause progressive i n f e c t i o n i n a group of twelve guinea p i g s . The same s t r a i n of organism, administered i n the same way as a l i v e vaccine to C h i n c h i l l a s , k i l l e d nineteen of twenty t e s t animals i n four weeks. The guinea pigs were not challenged w i t h v i r u l e n t s t r a i n s , but Gorham observes that the C h i n c h i l l a and not the guinea p i g s should be the t e s t animal f o r any vaccine t r i a l s . SPECIES [AFFECTED AUTHOR YEAR LOCATION DETAILS OF REPORTED OBSERVED OUTBREAK PATHOLOGY EPIZOOTIOLOGY CONCLUSIONS Silver Fox Rislakkiv 1942 Finland Infection tends to died out on a farm Dog Marten Claussens 1934 Germany? Mole Golovin 1930 Germany?. Unspecified Fur Bearers Marthedal 1954 1954 1961 Sweden Mink Knox-r-- Denmark 75 mink dead of unstated no Yellowish caseous nodules liver spleen kidneys & intestinal wall Due to feeding mink infected viscera of wild hares Mink Crews I 9 6 I Washington Only 2 animals Detail.not stated & Gorham western involved hut typical nodules state Organism seen isolated Chinchilla Chapman 1948 USA Chinchilla 1950 1954 Organism isolated by direct culture and guinea pig inoculation  Ranch overrun with mice & animals ran up on pens; eaten by mink at times Corvallis 1 isolation Not stated Oregon from chinchilla exactly, but No details nodules seen Chinchilla Leader & Baker Washington Died 2 days state USA after anorexia and depression Organism isolated often All livers with small yellowish white foci Histologically necrotic foci from nodules in w i t h lymphocyte affected livers & macrophage zone around V. Little encapsulation & giant cells Premises were contaminated by wild rodents & exchange of animals between premises continued overleaf TABLE XIX PSEUDOTUBERCULOSIS IN CAPTIVE FUR BEARING ANIMALS SOME.REPORTS BY VARIOUS AUTHORS o SPECIES A.FFECTED AUTHOR YEAR LOCATIOI DETAILS OE REPORTED OBSERVED OUTBREAK PATHOLOGY EPIZOOTIOLOGY CONCLUSIONS Ch i n c h i l l a Benito & Borrel 1957 France Nodules l i v e r & spleen Haem e n t e r i t i s , with multi necrotic f o c i i n colon. Enlarged C h i n c h i l l a Bankier & Langford 1959 to B r i t i s h Columbia Mod to severe losses i n 8 Similar to rpt of Leader & Usually exchange of infected 1962 -premises.Some repeats. Baker animals between premises Nutria Pallaske 1933 Leipzig 2 natural infections w i t h 1 i s o l a t i o n s Nutria • R i s l a k k i 19*2 Finland At least one outbreak.on a farm Nutria Wozniak 1958 Varsova & Chwalibog Nutria P i l e t c et a l . France 5 dead of 5G,G Emaciation Anorexia Depression Nodules i n lungs c h i e f l y . Also spleen, kidneys, l i v e r 2nd outbreak: Diarrhoea symptoms outbreak controlled by i s o l a t i o n of affected animals and treatment with parenteral Oxytetracycline Lobar pneumonia Acute E n t e r i t i s TABLE X I X PSEUDOTUBERCULOSIS IN CAPTIVE FUR BEARING ANIMALS page 2 SOME REPORTS BY VARIOUS AUTHORS 1 5 2 (b) Hares and Wild Rabbits Judging by the number of r e p o r t s , the f i n d i n g of P a s t e u r e l l a pseudotuberculosis I n f e c t i o n i n hares and perhaps w i l d r a b b i t s , i s common i n Con t i n e n t a l Europe and p o s s i b l y Scandinavia. Apparently Lerche i n L e i p z i g (1927) was the f i r s t to mention hares when comparing rodent i n f e c t i o n s to those of tur k e y s . P a l l a s k e (1932) s t u d i e d histopathology i n the hare us i n g Lerche's m a t e r i a l . In 1938" p i t reported an 'epidemic' i n hares i n Germany. From I t a l y came a report by Avanzi (19^8) °^ P a s t e u r e l l a pseudotuberculosis being i s o l a t e d from a hare i n Tuscany, and l a t e r , from S l o v e n i a , R i g l e r (195^) and Stefanovic (1957) reported s i m i l a r f i n d i n g s . In France Boquet (1937)> V a u t r i n (19^9), and Goyon (1956) c o n t r i b u t e d to the l i t e r a t u r e i n t h i s regard. Goyon s t u d i e d the b a c t e r i o l o g y and serology of ten s t r a i n s so i s o l a t e d . From Sweden Thai (195^) reported on the immunologic c h a r a c t e r i s t i c s of 186 s t r a i n s of P a s t e u r e l l a  pseudotuberculosis many of them ofSwedish o r i g i n . I t i s note-worthy t h a t no l e s s than ni n e t y - t h r e e of these s t r a i n s were from hares and r a b b i t s . This was more than f o u r times the number a t t r i b u t e d to the beaver, the second on the l i s t of mammalian c o n t r i b u t o r s ; and almost f i v e times the number 1 i s o l a t e d from turkeys, the highest avian c o n t r i b u t o r . S o l t y s 1 At a l a t e r date, Thai (1961) s t a t e d t h a t between 1948 and i 9 6 0 some 154 ( 6 . 2 per cent) of 25OO hares^examined i n Sweden were found to be i n f e c t e d w i t h P a s t e u r e l l a pseudo-t u b e r c u l o s i s . "~ 1 ~ 153 (194-7) found infection with Pasteurella pseudotuberculosis in a high percentage of hares in Scotland while conducting a survey for Tularaemia. Other reports have been published by Weiden-muller (1959) in Germany and Knox-Seith ( I 9 6 l ) in Denmark. The report by the latter on Infection of mink due to feeding hare viscera has already been mentioned. (c) Wild Mammals other than the Hare In view of the broad range of host susceptibility, the few reports in Table XX seem sparse Indeed. It i s well accepted that Pasteurella pseudotuberculosis is capable of causing sweeping epizootics among wild rodents and birds. A glance into the literature on plague gives ample Justification for the assumption that many such epizootics of sylvatic plague are completely unobserved and are l i k e l y often undiscovered. Doubtless as time goes on and more work Is done in population dynamics, a more accurate picture may be pieced together con-cerning epizootics of pseudotuberculosis. Table )£XX^ includes two previously unreported in-volvements of beaver in Canada with the possibility in one oase that the epizootic of pseudotuberculosis (which is assumed) may have also involved muskrats in the same area, (Queen Charlotte Islands). AUTHOR YEAR SPECIES LOCATION DETAILS Keymer 1959 Wild rodents i n general England The disease i s common among wild rodents which may spread i t to captive hardbilled birds by contamination of the seed We :i d e r i m u l l e r 1959 F i e l d mice and house mice Germany The organism was found i n faeces of ten of t h i r t y mice ( f i e l d mice and house mice). Mice were regarded as the chief c a r r i e r s of i n f e c t i o n Haas • 1938 Rat USA A s t r a i n was studied.which was recovered from a rat Risla k k i 19^2 Beaver Finland Not apparently i n epizootic proportions Bankier and Humphries . 1952 Beaver Muskrat? Queen Charlotte Islands Canada An i s o l a t e d dead beaver found i n an.area where both beaver and muskrats had died off. Presence of numerous discrete caseo-necrotic nodules i n l i v e r , spleen, l i v e r and kidney. Pasteurella pseudotuberculosis' i s o l a t e d and i d e n t i f i e d Langford 1954 Beaver Jasper Park Canada . Epizootic.of suspected tularaemia...Two beaver received. Pasteurella pseudo-tuberculosis i s o l a t e d i n both instances. Rabbits were k i l l e d with ease by IP: i n j e c t i o n of strains i s o l a t e d Claussen 1934 Muskrat Germany No d e t a i l s obtained TABLE XX SEVERAL REPORTS OF PASTEURELLA PSEUDOTUBERCULOSIS INFECTION OCCURRING IN.WILD MAMMALS 155 Pseudotuberculosis in Domestic Animals (a) Farm Animals . \ Table>XXI l i s t s a number of references to pseudo-tuberculosis infection in sheep, cows, horses, goats and the pig. Since a l l these animals graze, with the possible exception of the pig, one would expect them to be unavoidably in contact with the excretions or degenerated carcasses of birds and rodents which have been Involved In epizootic or sporadic infections. One can expeot the level of reported incidence of pseudotuberculosis to parallel the degree of use of laboratory diagnostic f a c i l i t i e s , since even provisional diagnoses of this condition are seldom i f ever made in the f i e l d . In light of this, one must assume that the figures are probably not representative of the degree of Involvement of farm animals. It is interesting that Knapp (1959) raises the possibility of human infection from one source of contact with cows. When this author (Stovell 1953) obtained an isolation from a bovine mesenteric gland, no other references to cattle could be found on a preliminary literature search. It was f e l t , and s t i l l i s , that a survey of diseased mesenteric glands from this species should be carried out before this finding deserves a report per se. AUTHOR YEAR LOCATION SPECIES DETAILS Murray 1932 Australia Sheep• Isolation of B. pseudotuberculosis (rodentium) from sheep' Truche 1938 Australia Sheep Identified a strain of P. pseudotuberculosis isolated . bv Gilruth (quoted by Jamieson Sol.ivs, 19*7) Jamison & Soltys 19*7 Scotland Sheep (rams) . Epididymo-orchitis'of rams associated with P.- pseudo type IB. 3 lambs with infection limited to testicle and epididymus. Tick activity was marked (July & August) infected hares in area Placidi et al 19 51 Morocco Sheep Details not obtained Watson 1962 England Sheep P. pseudotuberculosis has been isolated from ovine foeti on six occasions since i 9 6 0 Boquet . 1937 France Cow Mentions infection in this species (Atter Nocard' I889) Mazzini 1897 Italy? Cow After Popp'e (1928) cited by Knapp (1959) Stovell 1953 BC,Canada Sow Organism isolated frommesenteric lymph node with caseo necrotic lesions suspected of being tuberculosis strain •provisionally identified but culture lost Knapp (Graber & Knapp) 1959 Germany Cow Mastitis? Human (milker) with appendicitis form. . One cow with recently resolved purulent mastitis had ascending serum titre to 1:320. Other cows in area less"than 1:50. Pfeiffer 1890 Leipzig '-Horse Isolated from a horse suspected of having Glanders. Autopsy showed nasal and lung nodules with spleen,liver, and lymph nodes also involved Stephan 19*2 Horse Isolated three strains from horses Schlaffke 1921 German? Horse Isolated from a glanders-like'case in a horse Bauman 1927 Germany Goat Pseudotuberculosis in a young goat Ra,i agopalan 19** India Goat A case of Pseudotuberculosis in a goat Wramby & Frederison 19*1 Scandinavia Pig Described P. pseudotuberculosis infection in a pig TABLE XXI' PARTIAL DATA RELATING TO REPORTS OF PASTEURELLA PSEUDOTUBERCULOSIS IN FARM ANIMALS 157 (b) Cats and Dogs TableXXII l i s t s some of the l i t e r a t u r e references to i n f e c t i o n s i n these domestic animals. I t seems th a t s e v e r a l a u t h o r i t i e s r a t e the cat h i g h l y as a p o t e n t i a l source of human I n f e c t i o n s . The r e l a t i v e l y high r e p o r t e d incidence i n t h i s species i s not s u r p r i s i n g i n view of i t s d i e t a r y h a b i t s i n connection w i t h rodents, and the r e l a t i v e frequency of la b o r a t o r y d i a g n o s i s being c a r r i e d out i n the case of deaths i n pet cats? AUTHOR YEAR LOCATION SPECIES DETAILS Collet 1955 Prance' Dog Liver infection of dog with P. pseudotuberculosis Leblois 1920 Prance Cat Zoogleique pseudotuberculosis in the cat Cocu 1931 Prance Cat A case of Pseudotuberculosis in the cat Pallaske 1932 Leipzig Cat Used twelve spontaneous isolates from cats in Germany Verge. 1937 Prance Cat A case of Pseudotuberculosis in the cat Tottire-Hippoliti 19*2 Italy Cat Worked with two old strains from a cat Communal 19*5 Prance Cat Gives general description of infection in cats including personal observations on spontaneous cases Goret e_t al. 1957 Prance . Cat Describe laboratory diagnosis and pathogenesis in cats Winkle 1955 Germany Cat (in human disease) States '-cases in humans are very often due to . infected cats' Meyer ;" (in Dubos) " 1959? General Cats Comments that human infection has been attributed to cats TABLE XXII SOME REPORTS OP SPONTANEOUS PASTEURELLA PSEUDOTUBERCULOSIS INFECTION IN CATS AND A DOG H CO 159 Pseudotuberculosis i n Man U n t i l the p u b l i c a t i o n s of Masshoff and D d l l e (1953) human i n f e c t i o n s were proven i n but s i x t e e n cases. Of these, f i f t e e n cases ran a severe septicaemic t y p h o i d a l course with eleven ending f a t a l l y . F o l l o w i n g the observations of the above authors, a number of p u b l i c a t i o n s have appeared (mostly i n Europe) con-f i r m i n g the occurrence of mesenteric lymphadenitis i n humans wi t h a c l i n i c a l onset resembling that of acute or subacute a p p e n d i c i t i s . The c o n d i t i o n u s u a l l y a f f e c t s c h i l d r e n and young a d u l t s , p a r t i c u l a r l y males between the ages of three and twenty-t h r e e . A benign course w i t h uneventful recovery i s us u a l . The mesenteric a d e n i t i s i s u s u a l l y d e s c r i b e d as abscess-forming and r e t l c u l o c y t i c i n nature, (German l i t e r a t u r e r e p o r t s ) . I n North America the i n f e c t i o n has been diagnosed i n the San Francisco area (Goldman 1957) and more r e c e n t l y i n A l b e r t a (Hnatko and Roden 1962) . I s o l a t i o n of P a s t e u r e l l a pseudotuberculosis from the a f f e c t e d lymph nodes i s oft e n d i f f i c u l t ; however, since 195^ Knapp (1959) has e i t h e r through i s o l a t i o n s of the bacterium ( t h i r t e e n lymph nodes; two blood) or through s e r o l o g i c t e s t s ( n i n e t y - f o u r cases) and h i s t o l o g i c examinations, f u l l y proved the nature of the i n f e c t i o n . S e r o l o g i c a l t i t e r s of l.gO to l6o 1 1 : 1 2 , 800 have been recorded. The epidemiology i s considered by Meyer (1959) "to be obscure, although the vast animal reservoir with Its carriers and shedders is naturally suspect. Undoubtedly the route of entry is per oral as judged by the history of cases and con-firmed by the location of the focus of Infection. The wide avenue of possible contamination of human food i s well i l l u s -trated by the findings of Paterson and Cook ( 1962) . These authors incriminated f i e l d greens which were contaminated by pigeon droppings as the cause of recurrent pseudotuberculosis in guinea pig stock colonies. As mentioned earlier, in 1 9 6 l Daniels described the Isolation of Pasteurella pseudotuberculosis from the faeces of a patient suffering from pseudotuberculosis mesenteric adenitis. In addition the organism W&B also isolated from the faeces of a oanary belonging to the patient. 1 At the moment, i t is necessary, due to serological aberrations, to test patients' serum by use of the Widal test with live smooth antigen. 161 CARBOHYDRATE : AUTHOR*** 11* 15 16 17 18 19 20 21 22 A USUALLY REPORTED POSITIVE Arablnose + + + +d Galactose — + +d + + Glucose + • + + + + + + + G l y c e r o l +d + — + + Levulose + + + + + + Maltose + + + +d + +rt Marmitol -,. + + + + + + + Mannose + + + M e l i b i o s e Rhamnose + + + + + + S a l i c i n + + +v + + + Trehelose + + + + Xylose + + + +d B CONTROVERSIAL OR VARIABLE REACTIONS A d o n i t o l D e x t r i n - _+s I n u l i n - - -S o r b i t o l -Sucrose + - +* +r - + C - USUALLY REPORTED NEGATIVE A e s c u l i n Amygdalin D u l c i t o l E r y t h r i t o l I n o s i t o l Lactose M e l e z i t o s e R a f f i n o s e S t a r c h * on f i r s t i s o l a t i o n only *** r e f e r to Key on page d delayed r e a c t i o n r t room temperature s s l i g h t v v a r i a b l e r r e v e r t s ( a l k a l i n e ) TABLE X X I I I CONT'D CARBOHYDRATE FERMENTATIONS: REPORTED FERMENTATION WITH ACID NO GAS 1 6 2 CARBOHYDRATE AUTHOR*** 1 2 3 5 6 7 8 9 1 0 1 1 12 13 A USUALLY REPORTED POSITIVE A r a b l n o s e + + + + + + + + +10 + + + + G a l a c t o s e + + + + 1 1 + + + G l u c o s e + + + + + + + + + + + G l y c e r o l + + + + + + L e v u l o s e + + + + + + M a l t o s e + + + + + + + + + + M a n n i t o l + + + + + + + + + + + + Mannose + + M e l i b i o s e + + + + Rhamnose + + + + + + + + +10 + S a l i c i n + + + + + + a cbii.+ T r e h e l o s e + + + + X y l o s e + + + + + + + + + B , CONTROVERSIAL OR VARIABLE REACTIONS A d o n l t o l + _ D e x t r i n ± — + + + + I n u l i n - • — + -a + S o r b i t o l + - + + - -S u c r o s e — + + — — — + + — C • USUALLY REPORTED NEGATIVE -A e s c u l l n A m y g d alin + - -D u l c i t o l - - - - -a -Q -. - -E r y t h r i t o l - - - 11 -I n o s i t o l - - -L a c t o s e - - -M e l e z i t o s e + R a f f i n o s e - + — — - • , -10 S t a r c h 1 1 1 ** v a r i e s between 22 and 3 7 0 C i n c u b a t i o n r e f e r t o Key on page TABLE X X I I I CARBOHYDRATE FERMENTATIONS: REPORTED FERMENTATION WITH ACID NO GAS 1 6 3 BIOCHEMICAL. REACTION AUTHOR** Litmus M i l k R eaction I n d o l Production MR Test VP Test Production of H 2S Catalase Production Urea Decomposition N i t r i t e s Production Production of N H 3 from Peptone Methylene Blue Reduction Test G e l a t i n L i q u e f a c t i o n C i t r a t e Tests I n MacConkey Media B i l e Tolerance \% 10fo kOfo 37°C 7 day ( f i n a l ) pH i n glusose peptone water + + 2 3 4 5 6 sa sa a nc + + + + + + + +s + + + +a + + +st + + + 8 9 sa a + + + + + +-+s '+ + t +s + t 10 1 1 nc ^Claimed to be a d i f f e r e n t i a t i n g f e a t u r e from Pj. p e s t l s ( i n e r r o r ? ) **See Key. sa s l i g h t a l k a l i n i t y a a l k a l i n e formed nc no coagulation s slow st strong w weak TABLE XXIV BIOCHEMICAL REACTIONS AS REPORTED BY VARIOUS AUTHORS 164 KEY TO AUTHORS FOR TABLES XXI I I AND XXIV 1 Knapp (.1959) 2 Wilson and Mi l e s (1955) 3 Bergey (1957) 4 Meyer (1959) 5 Z i n s s e r (1957) .6 Devignat (195*0 7 Winkle (1955) 8 Zwick (1908) 9 S t o v e l l (1963) 10 Hadley (1918) 11 Beck and Huck (1925) 12 Lerche (1927) 13 Truche and Bauche (1929) 14 Lesbouyries ( 1 9 3 4 ) 15 Urbain and Nouvel (1957) 16 K i l i a n et a l . (1962) 17 Rosenwald and Dick i n s o n (1944) 18 Mathey and .Siddle (1954) 19 Moss and B a t t l e (1941) 20 Beaudette (1940) 21 Jamieson and S o l t y s (1947) 22 Wasielewski and Hoffman (1903) 165 BIOCHEMICAL REACTION AUTHOR** 12 13 14 15 16 17 18 19 20 21 Litmus M i l k Reaction I n d o l Production MR Test VP Test H 2S Production Catalase Production Urea Decomposition N i t r a t e s Reduction & N i t r a t e s Production NH-a Production from Peptone Methylene Blue Reduction Test G e l a t i n L i q u e f a c t i o n C i t r a t e Tests B i l e Tolerance \% k0% I n MacConkey Media 37°C 7 day ( f i n a l ) pH i n glucose peptone water a + + sa-only on + o r i g . i s , sa +w +S" +w +1 + ± *Claimed to he a d i f f e r e n t i a t i n g f e a t u r e from P_j_ pest i s **Se.e Key. sa s l i g h t a l k a l i n i t y a a l k a l i n e formed nc no coagulation s slow s t strong w weak TABLE XXIV CONT'D BIOCHEMICAL REACTIONS AS. REPORTED BY VARIOUS AUTHORS 

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