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The role of Sphaerophorus necrophorus in bovine hepatic abscesses Simon, Pullikattil Anna 1960

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THE ROLE OP SPHAEROPHORUS NECROPHORUS IN BOVINE HEPATIC ABSCESSES by PULLIKATTIL ANNA SIMON G.M.V.C., Madras Veterinary College (India) 1935 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF M.Sc. i n the Department of BACTERIOLOGY and IMMUNOLOGY. We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , I960. I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e - f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Bacteriology and Immunology, The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver 8, Canada. Date A p r i l 2&, 1960 - i i -ABSTRACT The purpose of this study was to find out the causative organism of bovine hepatic abscess; the character-istics of this etiological agent, particularly its immunogenic capacity, and to determine the degree of immunity conferred by vaccination of laboratory animals. Sixty-seven liver abscesses were collected from cattle at slaughter, from 5 5 of which Sphaerophorus necrophorus was isolated in pure culture. In another 7, i t was shown to be present in combin-ation with other organisms. The morphological, cultural and biochemical characteristics of this organism have been studied. The abilit ies of various media to support growth, form filaments, reduce lag phase, and increase the stationary phase, have been studied. The effects upon growth of other contributory factors, such as dose of inoculum, age of the seed culture, pH of the medium, temperature of incubation have also been investigated. An attempt was made to immunize 1 3 rabbits and 7 guinea pigs by inoculating kil led organisms. When challenged by the intravenous route with living culture, a l l the vaccinated animals resisted M.L.D., but succumbed to 1 5 . In rabbits, intravenous Inoculations of washed suspension of the organism produced a higher titre of agglutinins than the subcutaneous inoculations of heated whole culture. Rabbits with tltres of 1 : 2 0 , 0 0 0 did not show any more immunity than those with 1 : 2 5 0 0 . - i i i -The sera of cattle with hepatic abscess showed in general slightly higher agglutination titres than those free from abscess. However, this difference was on the whole slight. Hence, it is concluded that the agglutination test cannot be employed in the diagnosis of necrobacillosls. Judging by the cross-agglutinability, and despite some evidence of complex agglutinogenic structure, i t was concluded that the various strains studied belong to one fair ly homogeneous group. Two types of agglutinations, granular and floecular, were found to occur, depending on the average length and age of the organism used in the test. It was observed that the site of coherence which may correspond with the site of adsorption of the agglutinins is situated terminally or subterminally in both filaments and rods. iv -TABLE OF CONTENTS Page No. I INTRODUCTION 1 II DEFINITIONS 6 III HISTORY 7 IV TAXONOMY 17 V MORPHOLOGY AND STAINING CHARACTERISTICS 20 VI MEDIA AND METHODS 2k 1 . Introduction 2k 2 . Current Observations (Liquid Medium) 26 3 . Current Observations (Solid Medium) 29 k. Method of Preparation 3 1 5 . Preliminary,Experiments 33 VII BIOCHEMICAL REACTIONS kj, VIII PATHOLOGY 44 A. Cattle 50 B. Horses 59 G. Swine 60 D. Mink 6 l E. Fowls 6 l F. Rabbits 6 l (a) Introduction 6 l (b) Current Observations 62 G. Man 64-I X IMMUNOLOGY 66 A . Object 67 B. Materials 67 - V -Page No. C. Preliminary Experiments (0~ D. Immunization Procedure 72 X DISCUSSION 86 XI SUMMARY gg XII APPENDICES 91 XIII BIBLIOGRAPHY S& TABLES 1. Condemnation of Livers (Canada Packers) 1 2. Condemnation of Livers ( In Canada) 3 3- Growth in Different Media 3* k. Influence of Inoculum 37 5 . pH Changes Due to Autoclavlng 39 6. pH Ranges 39 7- pH Changes In The Medium 4-0 g. Relationship of Temperature 4-1 9. Lesions In Rabbits 63 10. Details of Routes and Vaccines 73 n . Agglutinin Titres of Immunized Animals 7^ 12. Cross Agglutination 76 13. Agglutination Reaction of Sera from Cattle with Hepatic Abscess 77 14-. Agglutination Reaction of Sera of Cattle Free from Abscess 77 15- Determination of M.L.D. gl 16. Challenge of Immunized Animals g2 17. Determination of M.L.D. g3 l g . Second Challenge g4-v l -Page No. PLATES 1. Fifth day Reaction in Rabbits 110 2. Eighth day Reaction in Rabbits 110 3. Fourteenth day Reaction in Rabbits 111 k. Post-mortem Lesions 111 5. Eighteenth Hour Culture 112 6. Agglutination Reaction Showing Rectang-ular Formation 112 7. Agglutination Reaction Showing Point Attachments 113 g. Agglutination Reaction In Tubes Showing Floccular Reaction 113 - v i i -ACKNOWLEDGEMENT I would like to use this opportunity to express my gratitude to Dr. G.E. Dolman, Head of the Depart-ment of Bacteriology and Immunology, without whose encouragement and guidance, this work would never have been undertaken or completed. My thanks are due to Dr. P.L. Stovell, Head of the Federal Animal Pathology Research Labora-tory, Western Branch,for the materials and fac i l i t ies . Among my co-workers I have to thank Mrs. E.M. Boivin, and Miss Dale Young for assist-ance in preparation of media and Miss Barbara Coles for photography. - 1 -CHAPTER I. INTRODUCTION During the oouree of employment as a Veterinary Meat Inspector of the Federal Government, the author had occa-sion to condemn, on account of abscesses, nearly 50$ of l i v e r s of c a t t l e slaughtered from some feed l o t s . This indicated the economic importance of the problem. There i s a common b e l i e f among the packing house and feed-lot operators that abscess of l i v e r i s an unavoidable consequence of fattening c a t t l e . Many early workers i n the f i e l d of bacteriology found these abscess-es s t e r i l e (at l e a s t , according to t h e i r methods of culture at the time). Text books on meat inspection f a i l to throw l i g h t on these questions. The table below I l l u s t r a t e s the condemnations of l i v e r s of c a t t l e k i l l e d i n Canada Packers, Vancouver, during 1 9 5 S . Table 1. Condemnation of Livers \Canada Packers) Period of slaughter Total No. of ca t t l e k i l l e d L i vers condemned due to abscesses(a) Per cent condemned Jan.-March 7,222 1,537 21.23 Apr.-June 5,722 1,216 21.25 July-Sept. 7A90 1,52^  20.34-Oct.-Dec. 1 2,350 1,54-S lg.53 (a) These do not include condemnations on account of angiomatosis or adhesions. The t o t a l number of c a t t l e slaughtered i n Canada and the number of portions (most of which are l i v e r s ) condemned on account of abscesses, f o r the years 19^4 to 1 9 a s appear-ing i n the Annual Reports of the Veterinary Director General are furnished i n Table 2 on the next page. (Angiomatosis, being a predisposing factor, and adhesions, a c o r o l l a r y of either abscess formation or abscess resolution, are included in t h i s t a b l e ) . TABLE 2. Condemnations Of Livers In Canada ^ Year i- ... ' Cattle Slaughtered Portions condemned f o r - Tot a l Percent Abscess Angiomatosis Adhesions Condemned Condemned 1 9 ^ 1,107,57^ ... 106,210 9,^99 6.1,785 177, W 16,0 194-5 1,4-81,800 121,185 12,598 83,965 217,7^8 1^,7 194-6 1,801,112 15 ,^4-10 15,570 9^,776 26^,756 1^7 19^7 1,551,5*58 140,74-1 13,097 72,771 226,609 1^.6 194-8 1,352,793 136,737 12, W 65,274 2l4-,505 15.8 19*9 1,^61,899 150,124- 15,297 80,4-56 2^6,4-77 16.8 1959 1,W,70^ 1^1,171 18,583 90,94-2 250,696 17.3 1951 1,227,975 119,999 18,166 89,443; 227,608 13,5 1952 1,121,692 121,902 13,167 7 M 3 3 209,202 18.6 1953 1,335,928 138,656 11 ,3^ 3 3 , 3 ^ 23^,02^ 17r5 195^ 1,521,936 1*7,&&7 1^,103 100,317 262,307 17.2 1955 1,64-5,27.6 176,1*16 16,787 117,653 3510,856 1S?9 19,56 1,740,117 208,74-5 18,116 130,54-8 357 ,^9 20.5 1957 1 ,895 , 7^ 265,55^ 16,313 136,896 ^18,763 22.1 1956 1,999,169 298,589 16,180 14-5,035 4-59,804- 23.0 The c o l o s s a l waste can be better appreciated when one r e a l i z e s that the 4-59,SC4 l i v e r s condemned i n 19 5& are from the 18>9 Federally-inspected slaughter-houses and that there i s an equal number uninspected. Thus the average annual loss can be estimated i n the neighborhood of 2-£ m i l l i o n d o l l a r s , computing each l i v e r as valued at $5.00. Apart from t h i s loss of l i v e r s , there i s consider-able loss to the feeder because of d e f i c i e n t feed u t i l i z a t i o n and weight increase. An occasional death i n the herd due to acute hepatic abscess i s not uncommon. This s i t u a t i o n i s not p e c u l i a r to Canada. Jensen ((1953) states that i n Denver the incidence of l i v e r abscess i s lk% of a l l c a t t l e slaughtered and the t o t a l annual loss i s $3:00,000. Frederick ( l$kj) states that the annual loss to the U.S.A. i s $1S,209,^6 per year. The disease has a universal d i s t r i b u t i o n , but shows high incidence i n c e r t a i n areas and low i n others. In one feed l o t v i s i t e d i n Kelowna, the incidence was &0% . Out of 337 head of beef c a t t l e slaughtered from a feed l o t at Calgary during 1959, 72% showed l i v e r abscesses. ( Avery 1959) . In the absence of s u f f i c i e n t data to suggest pre-ventive measures, a more c r i t i c a l and exhaustive study i s necessary to decide:- ' 1 . What i s the causative organism? 2. Does t h i s organism produce antibodies i n the system of the host? 3". Does i t produce antibodies i n s u f f i c i e n t numbers to Impart immunity? 4-. Can immunity be set up a r t i f i c i a l l y by vaccination? A number of subsidiary questions w i l l be dealt i n general as f a r as i s necessary. 6-CHAPTER II. D e f i n i t i o n s 1. Hepatic abscess of c a t t l e i s an in f e c t i o u s , non-contagious, acute or chronic necrosis of the l i v e r , characterized by single or multiple f o c i of variously coloured, f o u l smelling pus contained i n an encapsul-ated, nonvascular, f i b r bus. tissue. The usual causal microorganism i s Sphaerophorus necrophorus. Sometimes the disease i s c a l l e d n e c r o b a c i l l o s i s . 2. Sphaerophorus necrophorus i s a Gram-negative, anaero-bic jnonmotile, nonsporulating, nonflagellated, pleo-morphic organism, that may vary from coccoid forms to long filaments of 5°0 microns or more and causes necro-b a c i l l o s i s i n animals and human beings. 3. Vaccine The suspension of k i l l e d bacteria used f o r inoculation for_the purpose of immunization w i l l be c a l l e d a vaccine. CHAPTER III -7-HISTORY The h i s t o r i c a l aspect of t h i s problem can be dealt with under 2 sections, v i z . , hist o r y of bovine hepatic abscesses, and histo r y of Sphaerophorus necrophorus. A. History of Bovine Hepatic Abscess. Perhaps the p r o h i b i t i o n imposed on the use of l i v e r as part of the peace and burnt o f f e r i n g (Lev.3,^, and *J-,9). was i n recognition of the frequency of damage thi s organ suffered from abscesses. The e a r l i e s t among modern writers to recognize t h i s condition was MacFadyean who i n 1*591 re-corded cases of• ,disseminated Bliver of the ox and sheep. 190^  Mohler and Morse of the Bureau of Animal Industry, U.S. Department of Agriculture, brought the economic import-ance of l i v e r abscess to the public attention f o r the f i r s t time. 193° Orcutt,Marion L. i s o l a t e d the organism from l i v e r abscess i n cows and studied morphological, c u l t u r a l and serolog-i c a l characters. 1933 McGregor demonstrated for the f i r s t time that Sphaero- phorus neorophorus i s responsible f o r l i v e r abscesses. 1932 Newsom made a study of one hundred bovine l i v e r abscesses to determine the causative organism and found Sphaero-phorus necrophorus present i n $6% , In the same year Yamamato of Japan studied 17 bovine l i v e r abscesses and i s o l a t e d S. necrophorus from each. 194-2 Cooper and E l l i o t studied bovine and ovine metastatic abscesses. 194-3 Frederick reported the economic importance of the bovine l i v e r abscesses. 1944 H i l t o n Smith studied the r e l a t i o n s h i p between ulcers i n the rumen and abscess of l i v e r s i n c a t t l e . 194-6 Robert Getty studied the histopathology of f o c a l hepati-t i s . 194-7 Jensen et a l studied the abscesses of l i v e r s along with other conditions, such as angiomatosis, "sawdust" l i v e r and adhesions. 194-9 Madin made a b a o t e r i o l o g i e a l study of bovine l i v e r abscesses. 1951 Robinson conducted 2 experiments to f i n d out i f high nitrogenous d i e t was the cause of l i v e r abscess. 1954- Jensen et a l studied the r e l a t i o n s h i p between abscesses of the rumen and abscesses of the l i v e r s i n c a t t l e . 1956 Both J a r r e t and Searle Independently drew attention to bovine hepatic abscesses. 1957 Wostoupal wrote a review of n e c r o b a c i l l o s i s . These workers have established almost conclusively that Sphaerophorus necrophorus i s the primary organism seen i n bovine hepatic abscesses. But whether I t i s the primary factor or a secondary invader i s not decided. The p o s s i b i l i t y that d i e t i s a predisposing f a c t o r by causing undue s t r a i n on the l i v e r through excessive intake of a p a r t i c u l a r food ingredient or through a breakdown i n the defence mechanism due to some d i e t e t i c deficiency, remains to be investigated. B. History of Sphaerophorus necrophorus 1881 I t i s probable that the organism which Robert Koch found as bundles of wavy filaments, while examining histopath-o l o g i c a l sections of sheep cornea, was Sphaerophorus  necrophorus. lgglj. L o e f f l e r i d e n t i f i e d t h i s organism which he found In necrotic lesions i n the mouth of calves suffering from c a l f diphtheria (impetigo l a b i a l i s ) . He c a l l e d i t B a c i l l u s necrophorus. He succeeded i n producing necrotic lesions i n the mouse by subcutaneous inoculation of the d i p h t h e r i t i c membrane. He obtained primary cultures from mice on c a l f serum, but f a i l e d to sub-culture. Later he found i t in deep-seated tissues of calves suff-ering from navel i l l , i n necrotic lesions of the uterus, vagina, lungs and heart of cows. 1286 Flugge too found t h i s organism i n cases of c a l f diph-t h e r i a and c a l l e d i t c a l f diphtheria b a c i l l u s " ( B a c i l l u s  diphtheria v i t u l o r u m ) L a t e r he changed th i s name to B a c i l l u s necrophorus in agreement with L o e f f l e r . 1888 Shutz named i t on a morphological basis and c a l l e d i t the h a i r - l i k e or filamentous b a c i l l u s ( B a c i l l u s f i l l f o r - mls). 1889 Trevisan also observed i t In calves and oalled i t B a c i l l u s diphtheria vltulorum. 1890 Bang found th i s organism i n necrotic lesions of the snout and mouth of pigs s u f f e r i n g from the secondary - 1 0 -e n t e r i t i s , s t o m a t i t i s and pharyngitis of swine fever. He also found i t i n c a t t l e and horses suffering from foot rot, In calves s u f f e r i n g from navel i l l , and i n suppuration of kangaroos kept i n the zoo. He was able to i s o l a t e the organism from the i n t e s t i n a l contents of healthy hogs by rabbit inoculations; but was not successful with samples from the bovine. 1890 Widal and Nobecourt observed that pus from gangrenous tissue contained many bacteria demonstrable by d i r e c t smear; but on culture either very few organisms grew or none at a l l . 1891 Schmorl came across th i s organism i n a spontaneous enzootic among hi s laboratory rabbits, characterized by a spreading necrosis of the lower l i p . He inocu-lated mice with material from the rabbits and obtained cultures of the organism from the mice. He and his assistant acquired accidental Infections and each developed a finger abscess. 1891 MacPadyean saw t h i s organism i n "disseminated" necrosis of l i v e r s of c a t t l e and sheep. In 1900, he observed the organism to produce gangrenous dermatitus, necrotic lesions of feet, ulcerations of the intest i n e s and sec-ondary neorotic pneumonia i n c a t t l e . l&Sk- Gasperini, f i n d i n g the spreading filaments i n the sub-cutaneous tissue, c a l l e d i t burrowing fungus (Actino-myces c u n i c u l l ) . 1896 For the f i r s t time, Lehmann and Neumann encountered i t 11-l n calves. They thought i t to be a fungus and c a l l e d It c a l f diphtheria fungus (Oospora dlphtheriae v l t u l -orum) which name they changed three times. In 1899 they c a l l e d i t Actinomyces necrophorus, i n 1907 Coryne- bacterlum neorophorum, and i n 1927 Bacterium necro- phorum. 1898 V e l l l o n and Zuber, inspire d by the paradoxical obser-vation of Wldal and Nobecourt i n 1890, that many organ-isms were demonstrable i n gangrenous suppurations by d i r e c t smears and not by c u l t u r a l methods, conducted extensive research and from oases of suppurations i n human beings, i s o l a t e d a Gram-negative, nonmotlle, pointed rod, which often contained granules. It was anaerobic, and cultures produced a f o e t i d odour. Though not l e t h a l to rabbits and guinea pigs, i t pro-duced abscesses i n them. From the shape, they named this B a c i l l u s fuslformls. During the same year Jean Halle' in her researches on the bacteria of the female g e n i t a l i a , enoountered a s i m i l a r organism. She c a l l e d i t B a c i l l u s fundull- formls. Home recorded a case of foot i n f e c t i o n i n a reindeer from t h i s organism. 1903 Mettam observed i t i n necrotic lesions of calves, sheep and lambs. He was able to produce in rabbits by i n j e c t -ion, necrosis of the l i v e r , peritoneum, purulent a r t h r i -t i s , pneumonia with formation of abscesses, p l e u r i s y and p e r i c a r d i t i s . 12-1904- Basset produced an antiserum in the horse which pro-tected guinea pigs against f a t a l dose of the organism given i n t r a p e r i t o n e a l l y . (Many workers found guinea pigs naturally r e s i s t a n t to n e c r o b a c i l l o s i s . ) Mohler and Morse i s o l a t e d the organism during the same year from bovine l i v e r abscesses. Subsquently they i s o l a t e d i t ( i n 1905) from the normal alimentary canal of herblvora and (lnl910) from l i p and l e g ulcerations of sheep. 1906 Basset found i t caused a contagious f a t a l i n f e c t i o n i n rabbits commencing as necrotic ulcers, abscesses of the l i p , skin and subcutaneous ti s s u e . 1911 Reid found i t i n gangrenous foot rot of c a t t l e , f a c i a l dermatitis i n sheep, and n e c r o b a c i l l o s i s i n pigs. During the same year Gray found i t to cause stomatitis, and dermatitis of the feet of dogs. 1912 Ce'sari, alone and l a t e r j o i n t l y with Alleaux, studied th i s organism under the name " B a c l l l e de Schmorl" i n respect of pathogenicity, c u l t u r a l characters and immunogenicity. 1913 Bahr, by c a r e f u l l y regulated doses, produced antisera in goats and guinea pigs, which protected mice against l e t h a l doses of culture. During the same year Jensen Isolated S. necrophorus from the alimentary canal of healthy herblvora and pigs, and suppurative lesions i n kangaroos and monkeys i n the zoo. 1915 Cave found i t i n hepatic necrosis of unborn lambs and Mack found i t i n umbilical necrosis in lambs.two to. -13 -ten days old. 1918 Noleoheck encountered a serious outbreak of gangrenous dermatitis of horses at the U.S. Army camp at Hancock. Twenty per cent of the horses at the camp became i n f e c t -ed and 80% of those infected died. 1921 Cesari claimed to have immunized guinea pigs against the action of toxins produced by S. necrophorus,but d i d not have any great success i n protecting rabbits against l i v i n g organisms. 1923 Cesarl demonstrated the temporary immunity conferred by antiserum. He also studied the fermentation reactions of the organism on various carbohydrates. Balteano and U k i l also studied the fermentation reactions and v i a b i l i t y in a va r i e t y of culture media. Lignieres found i t as a secondary invader i n avian diphtheria and attempted passive immunization of rabbits with antiserum prepared from a horse. Though he was un-able to protect rabbits against test inoculations, he was able to protect them against natural i n f e c t i o n . Casper and Schumann, saw lesions s i m i l a r to c a l f diph-the r i a a f f e c t i n g adult c a t t l e i n epizootic form. 1924- Basset found the organism i n fowls as a secondary i n -vader i n avian diphtheria. Hagan, during the same year, i s o l a t e d It from bovine l i v e r abscesses and studied i t s v i a b i l i t y . He concl-uded that the production of H2O2 by the organism c o n t r i -buted to arrest i t s growth. He c a l l e d the organism Actinomyces necrophorus. l4-1924 G r i f f i t h also encountered this organism causing ab-omaeitis and death of c a t t l e in Nyasaland. 1926 Cameron and Williams found i t i n rabbits, causing the same Infection encountered by Schmorl and Basset e a r l i e r . Quinlan and associates recorded an outbreak which occurred i n 1923 in South A f r i c a among horses. F i f t y -three out of 130 were affected with gangrenous derma-t i t i s and 13 died with metastatic pneumonia. Two hundred and f i f t y c a t t l e and 40 donkeys remained free on the same premises. Turner found t h i s to be the cause of an outbreak of Black disease ( Infectious necrotic h e p a t i t i s ) of sheep i n A u s t r a l i a . 1933 Mettam and Carmichael found i t to cause f a t a l hoof infections of antelope i n A f r i c a . 1934 Beveridge studied the c u l t u r a l , morphological, bio-chemical characters of 11 strains of Sphaerophorus  necrophorus i s o l a t e d from bovine l i v e r s and other animals. He also studied i t s immunogenicIty,cross agglutination reactions, pathogenicity and oxygen i n t o l erance. He concluded that these are separate groups of stra i n s , some showing a rel a t i o n s h i p to each other and others with no relat i o n s h i p to any other. He also showed that t h i s organism can survive In atmospheric conditions i n symbiosis with aerobic organisms and that oxygen intolerance depends on the r e l a t i v e v e l o c i t i e s of peroxide formation and oatalase formation on the one 15-hand, and oxidation and reduction on the other. He showed that k i l l e d antigen d i d not confer immunity on experimental rabbits. Marsh and T u n i c l l f f i s o l a t e d Sphaerophorus necrophorus from cases of foot rot i n sheep In Montana. 1936 Feldman, Hester and Wherry measured the natur a l l y occur-r i n g S. necrophorus agglutinins i n the sera of various animals. 1933 Dack, Dragstedt,Johnson and McCullough Isolated what they c a l l e d Bacterium necrophorum from lesions of chronic u l c e r a t i v e c o l i t i s i n humans and l i v e r abscess-es i n bovines. They compared the morphological, c u l t u r -a l and pathogenic characters of these two groups. They could not f i n d any great difference except i n the great-er pathogenicity of the bovine strains f o r rabbits. T u n i c l l f f reported the persistance of s o i l i n f e c t i v i t y under natural subirrigated swamp pasture, ten months a f t e r sheep affected with foot rot had been removed from i t . He was not able to f i n d any organism a f t e r twenty months. 1933 Prevot while r e c l a s s i f y i n g the genus Bacteroldee of CosteHani and Chalmers created a new genus Sphero- phorus (Sphera (G-k) = Sphere; phorus = bearing,mean-ing "that which bears spheres" i n a l l u s i o n to the meta-chromatic granules usually seen i n thi s organism. The s p e c i f i c epithet necrophorus used by L o e f f l e r meant "death producing". However the generic name Sphero- phorus i s a homonym of Sphaerophorus persoon (1794), -16-a genus of lichens s t i l l being used by botanists (Fink, 1 9 3 5 ) , 194-5 Lahelle and Thjotta made a comparative study of fuso-bacterlum and "Necrobacterlum" with regard to t h e i r bio-l o g i c a l r e l a t i o n s h i p and proposed a new name for the l a t t e r . 194-7 Jensen, Frey, Cross and Cornwell studied the occur-rance of telangiectasis, "sawdust" and abscesses of l i v e r s i n feeder c a t t l e . They concluded that there i s a decrease i n l i v e r abscess incidence while i n feed l o t s . However t h e i r data indicate an Increase of 23% scars against a decrease of S% abscesses. As they d i d not take scar formation into consideration they f a i l e d to recognize the 1 6 ^ increase in abscesses during 167 days i n the feed l o t . (This w i l l be explain-ed i n the chapter on pathology). 1951 Robinson,Jasper and Guilbet showed that Sphaerophorus  necrophorus could survive extremes of rumen environ-ment. They f a i l e d to induce l i v e r damage by administ-ration of Sphaerophorus necrophorus, both by stomach tube and Intravenously. 1956 Prchal observed and recorded a case of cerebral abscess in a steer due to S. necrophorus. CHAPTER IV. -17 -Taxonomy V e i l l o n and Zuber i s o l a t e d many Gram-negative, non-sporing, anaerobic b a c i l l i , one of which they c a l l e d B a c i l l u s  f u s l f o r m l s . Later many workers saw fusiform organisms i n the mouth and i n the teeth of man and some animals.- ( T u n i c l i f f 1906, 1913, 1933; Ellerman 1907; Varney 1927; Slanetz and Rettger 1933; Bachmann and Gregor 1936; Pesch and Schmitz 193&; Spaulding and Rettger 1937; Hlne and Berry 1937; Kelly 1 9 4 4 ) . I t was Isolated also from the healthy alimentary canal of man by - Eggarth and Gagnon ( 1 9 3 3 ) ; Weiss and Rettger ( 1 9 3 7 ) ; Misra (1938) and Lewis and Rettger ( 1 9 4 0 ) . Relatively l i t t l e attention has been paid to the systematic study of these organisms. Bergey's Manual ( 1 9 5 7 ) nas placed these Gram-negative, nonsporing, anaerobic b a c i l l i with rounded or pointed ends i n the family Baoteroldaoeae, and divides these into 6 genera. In these 6 genera, 57 species are l i s t e d , as follows: Bacteroides 3 ° Fusobacterium 6 D i a l i s t e r 2 Sphaerophorus IS Streptobaoillus 1 Out of the l g i n the genus Sphaerophorus, 7 species are l i s t e d as nonmotile organisms that do not produce gas or liquefy g e l a t i n . The d i f f e r e n t i a t i n g characters available f o r these species are meager. The d i v i s i o n i s based on findings of one or two workers and the species have not been subjected to c r i t i c a l study. There i s no uniformity in the grouping of these species by d i f f e r e n t authors (see Bergey's Manual 1957, and Topley and - l g -Wilson 1 9 5 5 ) . Lahelle and ThJ0tta studied the rel a t i o n s h i p be-tween Fusobaoterium and S. necrophorus and stated that these should be placed as d i f f e r e n t genera of one family, Bacterla- ceae. They demonstrated that both these organisms have oommon morphological, biochemical, s e r o l o g i c a l and antigenic properties and antibodies. However, they d i f f e r e d i n t h e i r a b i l i t y to grow i n p l a i n peptone water, to ferment dextrose, and to hemolyse blood. Their main difference was i n t h e i r pathogenicity to laboratory animals. In reviewing the l i t e r a t u r e , one i s impressed by the lack of uniformity of methods employed i n the study of th i s organism, which makes i t more d i f f i c u l t to compare strains i s o l -ated by various workers. The m u l t i p l i c i t y of names used i s also evidence of the inadequate state of our knowledge concerning i t . The 6 t h e d i t i o n of Bergey's Manual quotes 16 synonyms; Wallis Hoare ( 1 9 1 5 ) , Hutyra and Marek ( 1 9 2 6 ) , Kelser and Schoen-lng (19^-g), Jordan and Burrows (194-9) and Merchant and Packer ( 1 9 5 6 ) c a l l i t by d i f f e r e n t names and quote more synonyms. Topley and Wilson (1955) oa.ll i t Fuslformis necrophorus and J u s t i f y t h e i r nomenclature on the p r i n c i p l e that a l l Fusiform species are not fusiform anymore than a l l Corynebacterium species are club shaped and a l l Clostridium species are spindle shaped. Jonsen and Thjgftta (194-g) suggested the binomial "Necrobacterlum necrophorus"which the Society of American Bacter-i o l o g i s t s rejected because of the difference i n gender of generic name Necrobacterlum and the s p e c i f i c epithet necrophorus. U n t i l - 1 9 -i t Is c o r r e c t l y c l a s s i f i e d and named.it may be better to leave i t where i t stands; therefore the name Sphaerophorus necrophorus w i l l be used i n th i s thesis in accordance with Bergey's Manual (1957). CHAPTER V. 20-Morphology and Staining Characteristics ( a) Introduction A l l workers have described S. necrophorus as pleo-morphic, forming long filaments, short rods, and coccoid bodies. No one observed m o t i l i t y except Schmorl (lg9l)who claims to have seen i t i n shorter elements. Ernst (1902) alone claims to have seen true branching. A l l writers agree that i t does not form spores, and f l a g e l l a have never been demonstrated. Ce'sari and Alleaux ( 1912) describe occasional Irregular forms showing fusiform and round enlargements at the ends. Other workers also have seen round swellings and elongated thickenings. (b) Current Observations During t h i s investigation pleomorphism was observed i n a l l s t r a i n s and cultures. Filaments longer than 300 microns were observed constantly, longer than 5 ° 0 microns frequently, and more than 1000 microns r a r e l y . Once a filament 1440 microns long was observed i n an eighteen hour culture i n Robertson's medium. Morphology was found to be influenced by the medium i n which It was grown and the age of the culture. In agreement with the findings of most workers but contrary to those of Henthorne ( 1 9 3 6 ) , more long filaments were seen In young abscesses and cultures than i n the old. In a r t i f i c i a l media, the long filaments break down into shorter rods more quickly than In lesions i n the animal body. In most media, long filaments break down into 21-shorter filaments or long rods i n 36-4g hours. With advance i n age, the filaments diminish i n number, so that i n cultures that are two weeks old, only few short ones may be seen. In Robert-son's medium without glucose, no filament i s seen after one week. In Modified Rosenow's Brain Broth (M.R.B.B) containing cystine and f e r r i c ammonium c i t r a t e , filaments as long as 5° o r more microns are seen a f t e r three weeks. In old abscesses, filaments are very rare; the common forms are coocold, as well as short and long rods. In s o l i d medium also, the breakdown of the filaments i s d i r e c t l y proportional to the age. In l i q u i d medium with good growth, the filaments w i l l be so numerous i n 18-24 hours that i t forms a matrix. The filaments are wavy with p a r a l l e l sides and r a r e l y stra i g h t ; but short rods and even long ones are mostly straight or s l i g h t l y curved. Sometimes a fusiform or round swelling i n the middle of the rod or filament may be seen. This i s peculiar to c e r t a i n strains and only a few organisms in a culture show t h i s feature. When seen, i t i s mostly observed In young cultures. The thickening i s about twice the breadth of the rest of the filament. In the rod form, the organism may be 0 .5 to 1.5 microns broad and 2-10 microns long. Some long filaments i n old cultures were only 0 .4 microns broad. The ends are rounded i n rods, and blunt-pointed i n growing filaments. Metachromatic granules are very c h a r a c t e r i s t i c of t h i s organism and are seen In cultures young and old. In a c t i v e l y growing young filaments i n cultures l e s s than 12 hours old, only one or two metachromatic granules are seen. In -22 -othera they may be so numerous as to give a beaded appearance. The d i s t r i b u t i o n of these granules i n the c e l l i s i r r e g u l a r both i n placing and spacing. They may be seen attached to one or both borders, or i n the centre. The shape also varies from spherical to Irregular. In old cultures, when filaments break down (which mostly happens at the s i t e of development of or proximal to the metachromatic granules), these granules are eith e r released as coccoid bodies or are found at the terminal points of the rods. This misleading phenomenon may have influenced Orcutt to believe that young cultures have more metachromatic granules than older ones. In a culture incubated at 4-5°C.it was found that most organisms developed many metachromatic granules before dying, thus i n d i c a t i n g that the c e l l was preparing to disrupt. At the time of d i s i n t e g r a t i o n of the c e l l these metachromatic granules may serve as a defence mechanism and a means of s u r v i -v a l during adversity or natural s e n i l i t y , b e i n g released and widely d i s t r i b u t e d as coccoid bodies. Spores or f l a g e l l a were never seen during t h i s i n v e s t i g a t i o n . No m o t i l i t y was observed i n short or long forms, nor i n young or old cultures. Probably Schmorl misinterpreted Brownlan movement as true m o t i l i t y . Very often short and long rods c l i n g together, giv i n g an appearance of branching. Under the d a r k f i e l d , when the cultures are examined without a cover-s l i p , a s l i g h t blowing on the wet f i l m w i l l cause enough move-ment to show that these are d i f f e r e n t organisms and not branch-ing forms. -23-The organism i s Gram-negative, non acid-fast, and can be stained by any a n i l i n e dye. Young cultures are r e a d i l y stainable while older cultures take stai n poorly. The metach-romatic granules take a deeper stain than the rest of the cytoplasm, which sometimes appears vacuolated. L o e f f l e r ' s methylene blue and d i l u t e carbol fuchsln p a r t i c u l a r l y bring out the beaded appearance. CHAPTER VI. Media and Methods 1. Introduction In general, anaerobic bacteria are more d i f f i c u l t to grow than the aerobic ones. Sphaerophorus necrophorus, though not ubiquitous, i t i s c e r t a i n l y found widely d i s t r i b u t e d i n nature. Yet very few organisms are more exacting i n t h e i r environment or fas t i d i o u s i n t h e i r dietary demands. Its v u l -n e r a b i l i t y to atmospheric oxygen and i t s wide d i s t r i b u t i o n are paradoxical. "The general lack of knowledge of fusiform  b a c t e r i a " wrote Slanetz et a l ( 1933) " i s d u e i n large measure to the extreme d i f f i c u l t y which has been encountered i n t h e i r I s o l a t i o n and c u l t i v a t i o n . " Many early workers i s o l a t e d S. necrophorus from lesions but f a i l e d to subculture. Mohler and Morse ( 1904) expressed d i f f i c u l t y i n inducing S. necro-phorus to grow. The contributions worthy of spec i a l notice are those of Von Hibler (1899); Lloyd (1916), and Cole and Lloyd ( 1917), Huntoon (1918), Haslam (1920), Hagan (1924), and Rosenow (193Q). Turner (1930) ,Beveridge (1934) and Prevot (1938) used V.F. broth and t h i s i s s t i l l being used by Prevot (1959) for c u l t i v a t i o n of S. necrophorus. Beveridge found Lepper and Martin's (1929) cooked heart medium, good f o r growth. However most workers f i n d that t h e i r cultures cannot be revived a f t e r a lapse of time ( T u n i c l i f f 1938-2, Dack et a l 193 !8 )>. This prompted T u n i c l i f f (1938-2) to f i n d a medium useful f o r storage. He modified Haslam's med-ium, and added l i q u i d p a r a f f i n as a s e a l . Though he claims to have kept the organism a l i v e f o r more than a year, t h i s has - 2 5 -not been corroborated by other workers. The p a r a f f i n seal makes the medium d i f f i c u l t to deal with. Broth Is rendered evenly turbid but clears In three to four days, leaving a d i r t y white chalky deposit (Cesari 1924) . Some strains grow as suspended p a r t i c l e s (Orcutt 1930) or as f l o c c u l a r deposit (Jensen 1913)• In l i q u i d serum and i n g e l a t i n medium, growth appears as f i n e f l o c c u l e s , which grad-u a l l y f a l l i to the bottom; the former i s not coagulated and the l a t t e r i s not digested (Ce'sari and Alleaux 1 9 1 2 ) . Nocard and Leclainche (1903) and Mohler and Morse ( 1 9 0 4 ) , however, found that l i q u i d serum was coagulated. Back et a l ( 1937) found two out of nine s t r a i n s digested g e l a t i n . Milk i s c l o t t e d i n 4- 6 days according to Cesari ( 1 9 2 4 ) , Orcutt (1930) and Henthorne ( 1 9 3 6 ) ; but not c l o t t e d according to Balte'ano and U k i l ( 1 9 2 3 ) , Kelser ( 1 9 2 7 ) , and Dack et a l ( I 9 3 7 h 0 1 t (1902) and Beveridge (1934) found growth in urine. With shake cultures, using deep medium ( 3 ° ° « c « per plate) of Huntoon's hormone agar and by establishing anaerobic conditions quiokly, Hagan (1924) obtained good growth i n 5£> "to 72 hours. The colonies were located i n the middle zone. Ernst (1902) Jensen (1913) and Orcutt ( 1 9 3 0 ) obtained growth i n deep agar tubes; but Quinlan et a l ( 1 9 2 6 ) f a i l e d to grow i t on p l a i n agar or g e l a t i n . Contrary to the reports of Hutyra and Marek, and Beveridge; Jonsen and ThJ0tta (1944) found potato extract favoured growth. Beveridge ( 1 9 3 4 ) found i t often d i f f i c u l t to obtain surface colonies on V.F. agar or serum V.F. agar. But -26-by using l e s s than 2k hour old V.F. agar plates, he obtained growth aft e r two days. On serum V.F. agar he obtained better growth. He also reports that once growth had been established under anaerobic conditions, i f the plates are incubated aero-b i o a l l y , growth w i l l continue f o r a few days. Dack et a l (193&) obtained growth on blood agar plates, incubated i n CO2 atmosphere, f o r t i f i e d with p y r o g a l l l c a c i d and sodium carbonate solution. He found that addition of 10$ sheep serum with 0.05 to 0.1$ cystine to the medium improved growth. Newsom (193*3) made cultures on rabbit blood agar plates and Cesari (192*0 on coagulated serum slopes. Most workers report growth on s o l i d medium as small colonies, 1 to 2 mm. i n diameter, c o l o r l e s s , opaque and d i r t y brown, of butyrous consistency. Jensen (1913) observed fuzzy colonies i n s o l i d medium, but Shaw ( 1933) noted that colonies were fuzzy on semisolid medium, and smooth on s o l i d medium con-tai n i n g 2% agar. Beveridge found that colonies i n i t i a t e d anaer-o b i c a l l y increased in size when incubated aerobically, the c r y s t a l c l e a r c o l o r l e s s colonies becoming opaque. 2. Current Observations Of C u l t u r a l Characteristics In Li q u i d Medium During t h i s investigation the following c u l t u r a l characters were observed. Cultures grew profusely i n M.R.B.B., Robertson's medium, and other l i q u i d media without anaerobic incubation, provided the media were heated and cooled just before inocula--27-t i o n , or were sealed a i r tig h t , while hot, a f t e r autoclaving. In 1% peptone water, growth was found to be very poor even when incubated anaerobically. In 2% peptone water with 1.5$ glucose, growth took place without anaerobic incubation when 1% inoculum from a 24-hour active culture was used. Acid and Gas Production Sl i g h t acid and gas i s produced by most strains i n M.R.B.B. and Robertson's medium, even when glucose i s not added. Sometimes i t may be as l a t e as 4-g hours before gas production i s seen, and i t i s never heavy. When 1% glucose i s added to the medium, gas production i s heavy. I t starts i n rare cases as early as g hours a f t e r inoculation and usually during 12 to 16 hours. Volumetrically the peak of gas production i s between l g and 36 hours; i t continues f o r 3 to 7 days and there may s t i l l be an occasional bubble a f t e r 10 days. This i s a sign of continued growth i n the medium. One s t r a i n caused such heavy gas production that a 25O ml. pyrex Erlenmeyer's f l a s k exploded in the Incubator i n 2^ hours. The f l a s k which was closed with an a i r tight screw cap, contained 200 ml. of M.R.B.B. with 1% glucose and was inoculated with half gram of pus from an abscess. When a t i g h t l y closed 2k-hour screw cap tube culture i s opened for the f i r s t time i n front of a flame, the gas explodes with a blue flame. In Robertson's medium containing 1% glucose, the gas production causes meat p a r t i c l e s to f l o a t on top of the broth. 2S-Turbldlty Broth i s rendered evenly turbid. During early stage of growth when gas production i s heavy, a i r bubbles can be obser-ved on the sides of tubes. In Robertson's medium, a f t e r 2 weeks incubation, the growth sediments, leaving the broth cl e a r at the top. A d i r t y brown deposit may be seen at the bottom of the tube at t h i s time. S l i g h t shaking w i l l disturb the deposit and make the medium turbid. After one month.turbidity i s r a r e l y observed even when the tube i s shaken. In Brewer's Thioglycollate Broth (B.T.B) the growth i s f l o c c u l a r and remains suspended u n t i l shaken, when i t gets evenly d i s t r i b u t e d . In 2% peptone water with 1.5$ gluoose, t u r b i d i t y i s evenly d i s t r i b u t e d . Colour The medium when i t contains 2.5$ f e r r i c ammonium c i t r a t e i s blackish. Growth renders i t p i t c h black. I f the i r o n , s a l t i s present at less than 0.23%, a black colour i s noticed at the bottom of the tube or as a deposit on the surface of the brain p a r t i c l e s . Odour Growth always produces a oharaoteristic, disagree-able, f e t i d odour, resembling that of rotten egg. The odour i s the same whether the growth i s i n the l i q u i d or s o l i d medium or i n animal t i s s u e . - 2 9 -35. Current Observations of Cul t u r a l Characteristics On So l i d Medium On s o l i d medium the best r e s u l t s were obtained when the medium was made up fresh or when plates were stored in carbon dioxide (C02). In plates exposed to the atmosphere f o r more than 2 days, the organism f a i l e d to grow. When It was grown i n l i v e r broth with meat mash and plated on l i v e r broth agar, growth was quicker. Twenty ml. of medium were used f o r a l l plate cultures. P l a t i n g was done by placing a drop from a Pasteur pipette on the medium. The drop was then touched once with an inoculating loop and streaked across, so that a thick f i l m of medium was l e f t at the s i t e where the drop was placed. This prevents complete drying up before anaerobic conditions are established. Not more than two plates were streaked at one time to avoid delay i n set t i n g up anaerobic conditions. For routine purposes, a l l cultures were incubated i n an atmosphere of p r a c t i c a l l y 100$ C02. To achieve t h i s , a Brown's anaerobic j a r i s evacuated by means of a water tap connected to a mercury manometer, (Gage manometer, combination pressure and vacuum model), u n t i l lg / l 9 t h s of the atmospheric a i r i s evacuated. The Jar Is then f i l l e d with C02 to atmospheric pressure and the process i s repeated twice. Usually growth appears i n hours, sometimes re-qu i r i n g 96 hours, as small (about 1 mm.) convex, c i r c u l a r , transparent g l i s t e n i n g colonies. When incubated f o r 2 weeks the colonies enlarge to about 3 mm. i n diameter and the centre - 3 0 -becomes opaque. Mostly the edges are regular in young colonies, becoming i r r e g u l a r when old. The consistency i s butyrous. Depending on the medium used, hemolysis Is complete or p a r t i a l and extends to about 1 m.m. from the edge of the colony. On prolonged Incubation, the surface becomes rough. Long f i l a -ments breakdown to long rods i n about 7 to 10 days. Growth always gives a f o u l odour. In shake cultures of brain heart infusion agar i n tubes, the colonies are very minute - of pin point size -i n 4-8-72 hours. Gas production i s so heavy that the medium becomes fractured i n various places. Further Incubation w i l l increase Isolated colonies to pin head s i z e . Surface colonies are not obtained i n plate cultures of brain heart infusion agar without blood. -31-4 - . Method of Preparation For t h i s investigation the media were prepared as follows: -Liver Broth. Thinly chopped pieces of c a t t l e l i v e r were boiled i n double the weight (by vol.) of water f o r l£ hours i n flowing steam. The supernatant was syphoned out and 1% peptone and 0.5$ NaCl. were added. The pH was raised from i t s usual l e v e l of 6A to using NaOH. It was then autoclaved for 20 minutes at 15 l b s . and stored. Brain. The covering membranes of fresh c a t t l e brains were peeled o f f and passed through a meat mincer with •| cm. cube cutter. Water equivalent to h a l f the weight (by vol.) of the brain was added, autoclaved at 3 l b s . f o r l j hours, and stored. At the time of making up the medium, the brain was strained o f f from the water and added to the l i v e r broth to make up nearly l / 3 r d the volume; 2.5 % CaCO^ and \% K2HPOIJ. were also added. ( This w i l l be c a l l e d hereafter the basic medium). For the purpose of comparing the influence of the d i f f e r e n t ingredients on growth, test materials were added to, or deleted from, the basic medium and growth was observed every k hours. Seven media were made up as follows:-Medlum No. 1 . To study the e f f e c t of f e r r i c ammonium c i t r a t e . Basic medium 100 ml. ge l a t i n 5 gm. glucose 1.0 gm. f e r r i c ammonium c i t r a t e 2.5 gm. Medium No. 2 To study the e f f e c t of absence of f e r r i c ammonium c i t r a t e . The same Ingredients as Medium Nol, - 3 2 -except that f e r r i c ammonium c i t r a t e was deleted. Medium No. 3 . To study the e f f e c t of absence of g e l a t i n . The same ingredients as Medium No. 1 , except that g e l a t i n was deleted. Medium No. k. To study the e f f e c t of maltose. The same ingredients as Medium No. 1 , except that maltose was added to make up a f i n a l strength of 1% a f t e r autoclaving. Medium No. 5. To study the e f f e c t of cystine. The same ingredients as medium No. 1 , exoept that 1 - cystine was added to make up a f i n a l strength of 0.1% a f t e r autoclaving. Medium No. 6, was Robertson's meat mash with 1% glucose. Medium No. 7» was tryptioase soy broth. The following s o l i d media were also used: -1 . Brain heart infusion agar without blood. 2. Brain heart infusion agar with 3% human c i t r a t e d blood. 3. Blood agar plates with 10% human c i t r a t e d blood. k. Blood agar with 3% bovine defibrinated blood. 5 . Blood agar with 10% bovine defibrinated blood. 6. L i v e r broth agar ( l i v e r broth as prepared for l i q u i d medium with 2% added agar.) The methods of anaerobiosis adopted were: 1 . Complete hydrogen atmosphere. 2. Complete COg atmosphere. 3. Ninety per cent hydrogen and 10% GO2 . -33-k. Ninety per cent C0 2 and 10$ H2 . 5 - P y r o g a l l i c acid and sodium carbonate solution. 5 - Preliminary Experiments  Experiment 1 To Study the S u i t a b i l i t y of Media f o r Growth  and M u l t i p l i c a t i o n of S. necrophorus In t h i s experiment the l i q u i d media numbers 1-7 were inoculated with a 2k hour culture i n M.R.B.B., using constant technique. Growth was observed by dark f i e l d micro-scopic examination every k hours. The s l i d e s were preserved, stained and examined l a t e r as a counter-check. (Because S. necrophorus mult i p l i e s by formation of long filaments, or by b l n i a r y f i s s i o n of short rods or by breakdown of long filaments into many short forms, i t i s necessary to take into consider-ation, not only the number of organisms per ml. but also the size of i n d i v i d u a l organism. This i s a necessary departure from the usual convention, where a l l organisms are p r a c t i c a l l y of equal lengths, and the term "growth" means an increase i n numbers; whereas, i n t h i s context, "growth" may mean only an increase i n length and not i n numbers. By multiplying the average length of the organisms seen In a f i e l d by the number present, a degree of quantitative p r o l i f e r a t i o n can be determined. This procedure was adopted wherever necessary to determine growth. The term "number of organisms per ml" means the number of organisms of an average length. The t o t a l length of the organism, when given, was obtained by multiplying the number of organisms by the average length). - 3 4 -Observations of the seven media l i s t e d were made for 10g hours. Since the results of growth i n media 1,2,5 6 are representative, and as the pattern of p r o l i f e r a t i o n a f t e r 4g hours i s much i n corroboration with that which preceded i t , r e s u l t s are furnished i n Table 3 below fo r those media noted above f o r a period of 4g hours of incubation. Table 3. Growth and M u l t i p l i c a t i o n of Organism i n Diff e r e n t Media Medium No>. Hours of Incubation Average length i n Microns Maximum Length In Microns No. per ml. 1 (with F e r r i c ammonium oi t r a t e ) 0 4 3 12 16 20 24 2g 3f g 15 12.g 10.4 g . l 13-5 10.g g . l 10.0 10.6 13.5 10. g 42 32.4 30.6 27.0 1 3 . 5 27.8 162.0 40-53 42.5 40.6 540.0 1.6 x 10| 2 x 102 2 x 103 109 10° 10© IQ7 5 Xl07a, 1 G g 10° 6 x io7 2 (without F e r r i c ammonium c i t r a t e ) 0 4 g 12 16 20 24 2& 15 7 12 20 & 42 4 3 2 2 42: 27 go 4oo 4255 420 450 g2 55 27 20 1.6 x i o | 1.6 x l o g 4 x 103 6 x i o § 2.2 x iol 3.3 x 1©' S.5 x 10' 5 x 1 0 ! g x 10Z 7.2 x 10X 3.4 x 10' (On. one occasion with a small dose of inoculum i n medium No. 1 a filament l440 microns long was seen a f t e r -35-l g hours of inoculation). Table 3 cont'd Growth and M u l t i p l i c a t i o n of Organism i n Diff e r e n t Media. Medium Ho. Hours of Incubation Average length i n Microns Maximum Length i n Microns No. per ml. (with cystine) (Robertson's medium) 0 k g 12 16 20 2^ 2g 21 0 k. g 12 16 20 2k 2g n 15 29.7 32.4 10. g 13.6 14. g 40.5 27.0 10.6 5.* 26 5* 81 40.5 2 7 , 5.* G C G k2 61 84-. 5 108.0 110.2 120.0 123.0 135.0 216.0 162.0 5^ -. 0 ^2 189 2^ 3 750 270 81 27 c c c 1.6 5 7 *.5 1.6 5 2 6 2 5.8 x X 4 i o | 10? 1 0 7 io7 10Z 107 10' io7 10 10 10 10^  107 10Z 1 0 g ii x 10 g (C means coccoid forms). Result of Experiment No. l It was observed that f e r r i c ammonium c i t r a t e increased the lag phase, and delayed breakdown and d i s i n t e g r a -t i o n . Absence of g e l a t i n d i d not adversely influence the growth. Maltose and cystine stimulated filament formation. They also delayed the breakdown of the filaments into smaller rods and coccoid forms. In general, cystine and f e r r i c ammon-ium c i t r a t e helped to maintain the v i a b i l i t y of the organism fo r longer period than any other combination, though the v i a -b i l i t y v a r i a t i o n was not phenomenal. In Robertson's medium, growth was quick with very l i t t l e l a g phase, and filament formation was excellent, but the breakdown w as equally quick. (One s t r a i n however remained without breaking down into coccoid forms or l o s i n g v i a b i l i t y f o r more than 4- weeks i n Robertson's medium). Growth i s poor i n th i s medium without glucose. In tryptlease soy broth, growth was very poor. For the purpose of oomparing the growth of the organism i n the laboratory medium with that i n the peritoneal ca v i t y of the mouse, 24- mice were inoculated l n t r a p e r i t o n e a l l y with 0 .1 ml. of a 24-hour culture. The mice were s a c r i f i c e d at 4—hourly i n t e r v a l s and the growth rate was assessed from the length of the filaments noticed In the peritoneal f l u i d . I t was observed that f o r g-12 hours the growth of the organism i n the peritoneum was very nearly the same as i n the medium containing cystine. Very long filaments over 100 microns were never seen i n mice and the breakdown was slow. Filaments as long as 5 ° microns were seen i n the peritoneum - 3 7 -4 days a f t e r the Injection. Among the media tested, medium No. 1 containing 1-cystine and 2.3% f e r r i c ammonium c i t r a t e was found to be the best. Experiment No. 2 To Study the Influenoe of Dose of  Inoculum on Lag and Growth Phale I t i s generally agreed that f o r good growth of S.necro- phorus a heavy inoculum i s necessary; but nobody has defined "heavy lnooulum" volumetrioally, In terms of percentage of the medium. Therefore to throw l i g h t on t h i s question, 3 test tubes containing 20 ml. each of medium No. 1 were inoculated with 3 drops, 1 drop and l / 6 t h of a drop, respectively, of a 24 hour culture i n M.R.B.B. The rate of growth, Judged from filament formation was noted every 4 hours and the r e s u l t s are tabulated below. Table 4 Influence of Quantity of Inoculum on Filament Formation Hours of Incubation Average length of filaments i n microns Maximum length of filaments i n microns 3 drops 1 drop l / 6 th drop 3 drops ldrop 1/6th droj 0 15 15 15 40 40 42 4 10 13.5 8.1 25 40 28 g 25 13.5 5.4 108 27 21 12 27 13.5 13.5 243 9 5 20 16 30 12 8.1 139 54 27 20 2g 12.8 12.6 216 108 27 24 15.8 13 10.8 135 82 135 23 14 8.1 8.2 94.5 82 108 10.g 9.5 150 io4 96 12.5 10.2 8.4 172 112 go 13.5 10.8 8.1 216 125 & 44 16 10.8 6.0 108 130 4g 4g 16 10.8 4.2 108 135 20 (Observations were made f o r 72 hours; but r e s u l t s are furnished for 4g hours, being representative of the entire -32 -period). The r e s u l t s indicate that large lnocula reduce la g phase and tend to produce long filaments. The average length of filaments was more i n the tube which received 3 drops of inoculum than i n those Inoculated with 1 or l / 6 t h of a drop. Filaments of maximum lengths of 2^ -3 microns were seen i n 12 hours In the medium which received the larger inoculum; whereas, filaments of only 135 microns maximum length were seen even a f t e r 2*J- hours i n the medium which received the le s s e r quantity of inoculum. Therefore i t can be concluded that a heavier inoculum promotes longer filaments. Though growth takes place i n a range of inocula from 0.003 to £5%, experience has shown that the optimum dosage i s 1% of the medium by volume. ( I f the seed culture i s old, th i s percentage w i l l have to be increased). Experiment 3 To Study the pH Range of the Medium  Suitable f o r Growth Before studying the pH range of the medium within which growth of the organism occurs, i t was necessary to note the pH changes which r e s u l t from autoclavlng medium No. 1 at 15 l b s . f o r 20 minutes. The findings are given i n Table 5 on the next page. - 3 9 -Table 5 pH Changes In Medium 1 due to Autoclaving pH of the medium before autoclaving pH of the medium a f t e r autoclaving 4.4 5 5 - 8 6 6.0 7 6.4 g 6.8 9 7-2 10 g.2 l l 9.0 (pH readings were taken on A.C. line-operated Beckman Model H 2 pH meter). To study the growth rate i n d i f f e r e n t pH ranges, a set of 6 tubes containing medium No. 1 and adjusted to pH 4.4 to 9 were Inoculated with a 24-hour culture. The results are tabulated below. Table 6 Comparative Total Length of Organisms i n Media at  Different pH Ranges Hours Total length of the organism per f i e l d at of Incubation PH 5 pH 6 pH 7 pH g 4 O.g 20 10 0.4 g 2.0 400 120 20 12 1 300 900 1 16 50 400 450 0.6 20 g 200 240 2 24 g 200 9go 30 2g 20 200 1000 go 3? 20 200 900 go 15 300 1280 go 40 10 450 1600 100 44 6 450 i4go go 4g 4 400 1260 60 (The t o t a l length of organisms per f i e l d i s obtained by - 4 0 -multiplying the average length of organisms i n a f i e l d by the number i n the f i e l d . Observations were made f o r lOg hours, but r e s u l t s f o r 4g hours only are furnished being representat-i v e ) . The table shows very good growth in terms of f i l a -ment length at pH 6 and 7 . Only considerably l e s s s a t i s f a c t o r y length was noticed at pH 8. Excepting f o r the 50 microns length seen at the l6t h hour of incubation, there i s nothing suggestive of growth at pH 5 . ( I t might be mentioned here that the Inoculum usually imparts to the medium a t o t a l length of 2 to 10 microns per f i e l d . The average length of the organ-isms In the inoculum was 15 microns and the maximum length was 4© microns - see the measurements at " 0 " hour i n Table 4 ) . There was no growth at pH 4 . 4 and 9 , hence i t i s not shown i n the table. Experiment 4 pH Changes i n the Medium Due to Growth Medium No. 1 at pH 7 was inoculated with a 24 hour culture and the pH changes were noted every 6 hours. The re s u l t s are tabulated below i n Table 7 ' Table 7 pH Changes i n the Medium pH of the Medium Before Inoculation No. of Hours of Incubal tion b 12 18 24 3O 3b 72 -7 7 6.8 6 . 3 6 . 3 6 .3 6 . 3 6 . 3 There was a lowering of the pH from 7 to 6 .3 -41-durlng the period of 6 to 18 hours a f t e r incubation began. This period coincides with the chief growth period. (See experiments No. 1 and 2 ) . Raising the pH from 6.3 to J &t k6 hours of incubation d i d not bring about new growth. Experiment 5 Influence of Temperature of Incubation on Growth" A set of 5 tubes containing medium No. 1 was i n -oculated and incubated at 22, 30, 37, 40 and 45° C. Growth was observed by d a r k f i e l d mioroscopic examination. I t was found that at 22 and 45°C. growth was not tangible. Hence r e s u l t s of growth at 30,37 and 40° are tabulated below. Table 8 Relationship of Temperature to Growth Hours of Incubation Total Length of Organism per F i e l d at Temper-atures i n Centigrade 3 ° 37 40 4 8 12 16 20 24 28 It 44 48 5P 300 900 750 300 300 450 450 150 150 90 60 120 800 600 500 1500 400 400 400 400 400 250 250 90 1500 1200 1200 1600 1000 300 400 360 450 400 360 Observations were made f o r 76 hours, but r e s u l t s are furnished f o r only 48 hours, being very c l o s e l y represent-ative) . -42-At 30°C. and at 40 C. growth was f a i r l y good. However, more metachromatic granules were seen at 40°C. than at either 37 or 3 0 . The thinness of the filaments was es p e c i a l l y noticeable at 40°C. and the organisms'were e a s i l y damaged. Growth was best at 37°C. Experiment 6 Influence of the Age of the Seed Inoculum Three tubes each containing 20 ml. of medium No.l were inoculated with 3 drops each from cultures i n M.R.B.B. medium 1 day, 1 week and 3 weeks old. The medium inoculated with 3-week-old seed culture took 72 hours to show growth and 120 hours to reach peak of growth, while those tubes inoculated with one day and 1-week-old seed cultures reached a peak of growth i n 24 and 4g hours respectively. When the dose of inoculum was raised from 3 drops to 30 drops of the 3-week-old culture, the peak of growth was reached i n 4g hours. CHAPTER VII. - 4 3 -Biochemical Reactions  Introduction Except f o r dextrose which a l l workers found to be s p l i t by S. necrophorus, there was very l i t t l e unanimity In the use of test substances or r e s u l t s . Addition of test material to d i f f e r e n t basic media tendsto Influence the reaction and i t s i n t e r p r e t a t i o n considerably, y i e l d i n g d i f f e r e n t r e s u l t s to d i f f e r e n t workers; eg. Harris and Brown (1927) and Balteano and U k l l (1923) found acid and gas production i n lactose while Orcutt (1930) Beveridge ( 1 9 3 4 ) and Henthorne et. a l (1936) found either acid or gas or none. The recorded reactions on other sugars, alcohols and glucosoides are likewise tinged with contradictory findings from one or the other worker. Current Observations Sugar fermentation reactions were tested by adding to 2% peptone water, a solution of the t e s t substance to make a f i n a l concentration of 1%. Acid and gas were produced i n dextrose, levulose, lactose and maltose; very s l i g h t acid and a f a i r amount of gas i n arablnose and g l y c e r o l ; no acid but s l i g h t gas In d u l c l t o l , galactose, i n u l i n , s a l i c i n , s o r b i t o l and trehalose. No a c i d or gas was produced i n mannitol, r a f f i n o s e and adonitol. Methylene blue was not reduced. The organism was found to be nitrate-negative and in d o l - p o s l t i v e . -44- -CHAPTER VIII. PATHOLOGY OF NECROBACILLOSIS Necrobacillosis has a widespread epizoology and has been recorded i n a l l continents of the world. 1 . Predisposing Factors Before considering pathogenic e f f e c t s of S. necro- phorus on the l i v e r , i t i s pertinent to consider the factors predisposing to l i v e r necrosis. Frey and Jensen (1946) deter-mined the vitamin A content of l i v e r s of c a t t l e slaughtered before,fattening and at i n t e r v a l s during fattening. They found that at the beginning of the fattening period the vitamin A content was high and that the l e v e l declined sharply i n the early stages of fattening. This period coincided with the time of abscess formation. In another experiment conducted by Frey, Jensen and Connell (1947), the vitamin content of the l i v e r was shown to have no dire c t bearing on abscess formation. Gyorgy (1957) found that necrosis develops a f t e r the experimental animals have become depleted of protective food constituents. I t appears that t h i s period of depletion i s prolonged by a n t i b i o t i c s , which, through the protection afforded by choline and methionine released through b a c t e r i a l d i s i n t e g r a t i o n i n the i n t e s t i n e , may be instrumental i n the prevention of f a t t y degeneration of l i v e r with consequent c i r r h o s i s . Toxio amines are formed i n the intestines from amino acids by the action of bacteria, Just as the pharmacolo-g i c a l l y i n e r t trlmethyl-amine i s formed, and the a n t i b a c t e r i a l agents temporarily i n h i b i t t h e i r formation (Nuttal et a l 1897). -45-I t i s most probable that ammonia and re l a t e d toxic substances found i n Increased amounts i n the blood i n hepatic coma are c h i e f l y formed through b a c t e r i a l action i n the i n t e s t i n e . They represent a tangible example of i n t e s t i n a l i n t o x i c a t i o n . In the presence of l i v e r diseases, I n t e s t i n a l b a c teria of saprophytic nature are often found i n the l i v e r and even i n the blood (Whipple;, and Harris 195°) • Vitamin E and a n t i b i o t i c s such as chlortetracycline and p e n i c i l l i n have been found to i n h i b i t the development of dietary necrosis of l i v e r i n r a t s . (Barness et a l 1 9 5 6 ) . On the other hand, removal of methionine and reduction of yeast from the d i e t , induced necrosis of the l i v e r within 60 hours i n r a t s , which were maintained previously on a depletory regimen f o r 50 days (Valberg and Beveridge i 9 6 0 ) . I t i s a well accepted theory, supported by many animal experiments, that the ruminal and i n t e s t -i n a l f l o r a a l t e r considerably with dietary change. The fa c t that under optimum conditions a s i g n i f i -cant f r a c t i o n of the t o t a l requirements of a known vitamin may originate from the ruminal f l o r a , i s not nearly as import-ant as the p o s s i b i l i t y that under disturbed conditions, the supply of an unknown factor which i s normally produced by the f l o r a i s reduced to a point where a conditioned deficiency develops. The synthetic fodder supposedly constituted to meet a l l the requirements of the animal "body, according to our present knowledge, may s t i l l lack an unknown factor, or may adversely influence i t s production through the symbiosis of ruminal bacteria. For the time being, there i s no d e f i n i t e -46-proof of the existance of such unknown factors normally furnish-ed by the ruminal f l o r a . Newsom (193*5) observed that the unusual prevalence of l i v e r abscess i n Colorado, Nebraska and C a l i f o r n i a , may be associated with the feeding of beet by-products. But the Univer-s i t y of I l l i n o i s launched an experiment to v e r i f y the v a l i d i t y of t h i s observation and found i t baseless. Frederick's i n v e s t i g -ation of the hypothesis that the disease was due to 'hogs follow-ing c a t t l e i n feed l o t s ' ended up with the same r e s u l t s . In an experiment to study the influence of excess protein in feed, c a t t l e were fed f o r 100 days on d i f f e r e n t concentrates of soy-bean meal, ranging from 12.5 to 33-33$ o f t h e i r feed. Only one l i v e r was condemned f o r abscess, and that i n the lower percent-age l e v e l group. During th e i r investigation the I l l i n o i s work-ers found that 9-2$ of the hepatic abscesses resulted from per-f o r a t i n g foreign bodies i n the stomach (Frederick, 1943). Robinson et a l (1951) also conducted two experiments to assess the claim that high nitrogenous feed caused l i v e r abscesses, and found no grounds f o r this assumption. Jensen et a l (1947) studied the Incidence of l i v e r abnormality of c a t t l e from feed l o t s . He recorded the experi-ence of the feed-lot-operators i n the following words, "It i s the common experience of the feed-lot-operators that c a t t l e from range take to concentrates slottfly, and the consumption Increases u n t i l about the 7°th day, when suddenly i t f a l l s nearly, by one-third. At no l a t e r time does i t increase. I t i s t h e i r experience that the maximum average d a i l y consumption -4 7-of concentrates occurs during the period of f o r t y to sixty days on feed." As a r e s u l t of his f i r s t experiment, out of the two he c a r r i e d out i n t h i s connection, he deduced that l i v e r abscesses develop i n many animals at approximately the 67th day, throwing them off feed and lowering the consumption rate. A f t e r h i s second experiment he concluded that there was no increase i n l i v e r abscess Incidence during the fattening period i n the feed l o t . The c a t t l e with which he experimented had a high incidence rate of 29$ , when they^were brought i n from the range. At the end of 167 days i n the feed l o t , t h i s f e l l to 20%. However, the data he furnished can be otherwise i n t e r -preted. His findings are reproduced i n the upper section of the table below. Days on Per cent Per cent Per cent Per cent Feed Telangiectasis Sawdust Abscess- Abscess scars 0 0 0 0 29 h i 10 0 10 16 77 40 0 5 25 120 37 5 16 21 167 35 15 25 20 Difference between pre-feed and post-feed period of 167 days + 25 -9 As shown by the section appended to the table, i f the scars and abscesses are added together, the table i n d i c -ates an addition of lS% at the end of 167 days. Many of the 29$ of the abscesses could have healed during t h i s period, - 4 g -undergoing soar formation, giving r i s e to 23% scars and 16% of other animals meanwhile developing fresh abscesses, the combined ef f e c t s of these processes r e s u l t i n g i n a 20% Incidence at the end of the period of observation. It i s possible that a high nitrogenous d i e t throws a s t r a i n on the l i v e r parenchyma rendering I t susceptible to i n f e c t i o n by S. necrophorus or other bacteria or viruses. When any tissue i s invaded by bacteria or viruses, anaerobic conditions may r e s u l t through c e l l destruction or normal oxygen consumption, creating a primary focus i n whioh S. necrophorus can s e t t l e down and p r o l i f e r a t e . A primary focus i s not always required. In heavy i n f e c t i o n s , when large numbers of organisms become embedded In one area, the endotoxin released by the destruction of some of the b a c t e r i a produces necrosis of the surrounding tissues, creating a focus f o r those organisms s t i l l v i a b l e . 2. Habitat The organism has been seen i n the normal aliment-ary canal of herblvora and i n the caeca of pigs. (Bang 1891, Mohler and Morse 1904, Ce'sarl and Alleux 1912, Jensen 1913). I t was seen i n the pasture s o i l of Montana 10 months afte r sheep with foot rot had grazed there, but was not found a f t e r 20 months. The organism survived one winter i n that rigorous climate. (Marsh et a l 193*0 • Though able to survive i n the s o i l for a considerable period, the capacity of the organism to p r o l i f e r a t e outside the body of i t s animal host has not -49-been established. I t f a i l e d to grow and multiply i n s t e r i l e s o i l and manure media ( T u n i c l i f f 1 9 3 8 - 1 ) . In nature, i t s a b i l i t y to survive may depend upon intimate association with aerobes (Beveridge 1 9 3 4 ) . In the animal body, esp e c i a l l y i n the tissues i t can survive f o r long periods. Freezing does not destroy i t i n six months. In frozen l i v e r abscesses and in frozen cultures 3. necrophorus remains viable f o r six months or more. Animals Affected The organism has been recorded as occurring i n a large number of pathological conditions i n 23 species of animals ( i n c l u d i n g birds and r e p t i l e s ) such as: horse, c a t t l e , b u f f a l o , sheep, deer, antelope, goat, dog, hog, fox, wallaby, monkey,mink, cat, rabbit, guinea pig, rat, mouse, buzzard, o s t r i c h , t o r t o i s e , snakes, and man. (Cesari,1912; Shaw,1933; Hagan,195l). I t was present i n a f i e l d - r a t found dead with necrosis of testes and abdominal muscles. In a l l Infections the primary manifestation i s necrosis. When the defence mechanism of the body succeeds i n walling o f f the i n f e c t i o n , an abscess forms. The disease i s designated d i f f e r e n t l y according to the animal and the tissues Involved. Apart from those disease e n t i t i e s s p e c i f i c a l l y designated as n e c r o b a c l l l o s l s , the organism i s seen i n ulcers, wounds, and as a secondary invader i n v i r a l and b a c t e r i a l i n f e c t i o n s . The most common species of animal affected i s the bovine, and the usual condition i t causes i s hepatic abscess. This condition can appear i n sporadic or enzootic form, and -50 -i n the l a t t e r may a f f e c t as many as 80% of the entire herd. In the form of foot rot or c a l f diphtheria i t may assume epizootic form. I t i s known to a f f e c t adult c a t t l e i n large numbers, producing lesions s i m i l a r to c a l f diphtheria (Casper and Schu-mann,1923). Isolated cases of omphalophlebitis i n calves may also occur. Occasionally i n horses i t produces gangrenous dermatitis In epizootic form. I t may also appear enzootlcally as necrosis of the hoof and heel of the horse and the antelope, associated with metastatic p l e u r i s y and f a t a l pneumonia. (Quinlan et a l , 1 9 2 6 ; Mettam and Garmichael,1933)• l a sheep, ulcerations of the l i p and l e g , f a c i a l dermatitis and foot rot, appear i n epizootic form, while necrotic h e p a t i t i s of lambs appears i n enzootic form.In rabbits, i t was found to cause widespread i n f e c t i o n . When i t appears i n fowls as avian diph-t h e r i a , the i n f e c t i o n spreads r a p i d l y around the flo c k . In other animals, i t has been found to cause only sporadic i n f e c t -ions. Some of the major diseases produoed by S. necro-phorus i n certain animal species w i l l be discussed below i n greater d e t a i l . However, th i s thesis w i l l be concerned mainly with infections i n c a t t l e and i n p a r t i c u l a r with bovine hepatic abscess. (A) Cattle In c a t t l e , S. necrophorus produces hepatic abscess-es, necrotic h e p a t i t i s , foot rot, necrotic dermatitis,necrotic rumenitis, abscesses of teat, and necrosis of uterus, vagina, alimentary canal, Joints and lungs. I t also causes c a l f diph-t h e r i a i n the form of necrotic stomatitis, g l o s s i t i s , or laryn-g i t i s . Route of Infeotlon For primary i n f e c t i o n S. necrophorus i s unable to penetrate the normal mucous membrane. Entry i s achieved through a break in the membrane, due to teething, or through wounds caused by castration. More commonly entry Is through abrasions i n the stomach and int e s t i n e s caused by awns and stalks of fodder, or m e t a l l i c objects i n fodder. Once entry has been achieved, i t s e t t l e s down i n any portion of the body and brings about necrosis. Having est-ablished i t s e l f , i t spreads to other organs by metastasis or through d i r e c t extension by i t s p r o l i f i c growth and a b i l i t y to penetrate the e p i t h e l i a l c e l l s , and even c a r t i l a g e s and tendons. Lesions i n the mouth of calves are mostly due to teething wounds. When abscesses are seen i n the v i s c e r a l sur-face of the l i v e r i n the region of the impresslo r e t i c u l a r i s , omasica or abomaslca, with adhesion between the concerned organ and the l i v e r , the route of entry maybe d i r e c t through a break i n the wall, caused very often by meta l l i c objects. I t i s not uncommon to f i n d the wall of the stomach or the S-shaped portion of the duodenum and the l i v e r , diaphragm and lungs, knit into one abscess. Robinson et a l (1951) f a i l e d to produce l i v e r abscesses in c a t t l e by intra j u g u l a r i n j e c t i o n of l i v e cultures. -52-Three out of l4 rabbits injeeted intravenously by the author did not show any l e s i o n i n the l i v e r , but instead developed lesions i n the lungs. One produced lesions also i n the brain. Long filaments of young cultures may become entangled i n the lung when administered intravenously through the jugular or a u r i c u l a r vein. Judging from the fact that long filaments were never encountered i n the c i r c u l a t o r y system, i t would appear that the coccoid forms or the smaller rods pass through the lung into the a r t e r i a l c i r c u l a t i o n and are di s t r i b u t e d . M i l i a r y abscesses Involving the entire abdominal vi s c e r a , diaphragm and lungs are then seen. Dose Abscesses can be produced r e a d i l y in experimental animals by i n j e c t i o n of via b l e cultures of S. necrophorus. The dose has a d i r e c t bearing on the i n i t i a t i o n of i n f e c t i o n , both natural and a r t i f i c i a l . During t h i s Investigation I t was ob-served that i n j e c t i o n s of 0.06 ml. of a 24 hour viable culture subcutaneously into rabbits f a i l e d to produce abscesses or death. In 2 out of 4 mice, 0.05 ml. of the same culture pro-duced lesions,and i n rabbits caused death i n 4—10 days when given In 0.1 ml. dosage subcutaneously. This observation coincides with those of Cesari and Beveridge. The pathogenic dose, however, varies with d i f f e r e n t s t r a i n s , with the number of generations and period of maintenance i n a r t i f i c i a l medium, the age of the culture and the i n d i v i d u a l s u s c e p t i b i l i t y of the rabbit. - 5 3 -1. Hepatic Abscess (a) Introduction This i s the most common form of n e c r o b a c l l l o s i s a f f e c t i n g large numbers of c a t t l e . I t brings about double economic loss to the packer, because he pays not only f o r the abscessed l i v e r which cannot be sold, but also f o r the abscess I t s e l f , which may weigh many pounds. Thereby he loses nearly $10'.per average animal. I t i s not u n l i k e l y that very soon, i f beef becomes more abundant, discrimination i n buying market may a r i s e , thus throwing the weight of the loss on the producer. The producer even now loses considerably through the poor feed u t i l i z a t i o n of the animal. Any derangement of the l i v e r - an important organ i n metabolism - i s d i r e c t l y r e f l e c t e d in the animalte a b i l i t y to u t i l i z e the feed and gain weight. In addition, an occasional loss of an animal due to toxaemia i s not uncommon. (b) Symptoms The c l i n i c a l manifestations of chronic hepatic n e c r o b a c l l l o s i s are r a r e l y more than s l i g h t anorexia, low fever, an arched back, a tendency to remain down, and reduced feed consumption and milk secretion. In acute cases terminating i n death, these symptoms are aggravated; the temperature may be between 104 and 106°F., r e s p i r a t i o n 30 and 40, and pulse 60 and 100. The animal i s constipated. I f the abscess i s i n the posterosuperior aspect of the l i v e r , to the l e f t of the oesoph-ageal notch, pain w i l l be evinced i f the region of the paralum-bar fossa posterior to the l a s t r i b i s palpated. Just before death, the animal may be struggling with abdominal pain. (c) G-ross Pathology Post-mortem lesions consist of one or more ab-scesses of sizes varying from 0.5 cm. to 4 cm. (In one animal an abscess was 24 cm. in diameter and 11 cm. thick, and when dissected out weighed l 4 pounds. Another animal had 159' abscess nodules covering the e n t i r e p a r i e t a l and v i s c e r a l surfaces, with more embedded in the parenchyma and not v i s i b l e f o r counting. Neither of these animals showed any premortem symptom nor any metastatic l e s i o n ) . Young lesions with very l i t t l e pus have a connect-ive tissue covering, in t e r l a c e d with f i b r i n bands and a central core of sequestered necrotic l i v e r t i s s u e . This core can be enucleated by pressing between the fi n g e r s . With age t h i s core disintegrates, becoming pus. The pus i s less viscous i n ol d abscesses than i n young, and yet i t can hardly be a s p i r -ated into a Pasteur pipette with a bore of 1.5 mm. diameter. Very frequently resorption commences within 3 months of the onset of abscess formation, and i n another 3 months only a scar tissue w i l l be l e f t to mark the s i t e . When mi l i a r y abscesses are seen, most of them are of the same si z e , i n d i c a t i n g a common source and period of i n f e c t i o n , while i n other cases i t i s usual to see abscesses of a l l sizes and ages on the same l i v e r . Abscesses situated within l i v e r tissue can be f e l t as s o l i d masses on palpation. -55-(&) Hlstopathology The c e n t r a l tissue shows coagulation necrosis. Microscopically, t h i s consists of a mass of leucocytes and "bands of fibrous tissue, necrosed c e l l s and abundance of S. necrophorus. At the periphery i s a pyogenic membrane which Is l i n e d on the inner aspect with f a t - f i l l e d mononuclear leucocytes and numerous lymphocytes, and on the outer, with a zone of fibrous connective tissue containing numerous plasma c e l l s . S. necrophorus organisms can be seen penetrating through t h i s encapsulation and reaching into the surrounding normal tissue. Sometimes other microorganisms such as strep-to c o c c i , staphylococci and corynebacteria are seen. Yeast c e l l s and 35. c o l l are r a r e l y encountered. ( e) Current Observations In some feed l o t s , the Incidence of l i v e r abscess i s high, while i n others i t Is low. Observations made during a v i s i t to 5 feed l o t s i n the i n t e r i o r of B.C. are stated below. In one of the feed l o t s at Kamloops, where c a t t l e are being fi n i s h e d f o r increasing the output of beef in compar-ison to l i v e weight, the incidence was n i l among the 50 c a t t l e slaughtered within the f i r s t 6 months of s t a r t i n g the project. Within the next 3 months the incidence was 4$. I t slowly rose during a period of nearly 2 years to 2&%. In another feed l o t the incidence was f l u c t u a t i n g between 30 and 50$. In a t h i r d (at Armstrong) i t was 40$ and i n a fourth (at Kelowna) i t was $0%. Avery (1959) reported a 72% incidence i n a feed l o t at Calgary. Marsh et a l (193*0 have shown that an infected - 5 6 -pasture can r e t a i n i n f e c t i o n . In the feed l o t with an incidence of S0%, there was foot rot among the c a t t l e . So f a r , investiga-tors have concerned themselves with the feed, the nitrogenous r a t i o , and the d i f f e r e n t ingredients that were l i k e l y to cause abscesses. The problem of crowding and lns a n i t a t l o n has eluded s p e c i f i c investigation. Crowding has a di r e c t bearing In that the organ-isms from the b i l e of c a t t l e affected with l i v e r abscess are discharged through the b i l e into the intestines and the feces, causing contamination of the fodder. Those animals unable to reach the trough w i l l pick up whatever has been s p i l t on to the ground. Overcrowding, insanitary housing conditions and i n c l e -ment weather contribute to heavier contamination of the fodder. "The accumulation of manure, aided by several weeks of warm wet weather, formed a natural incubator f o r B a c i l l u s necrophorus" wrote Kolecheck ( 1 9 1 8 - 1 9 ) . This cyole of excretion and contam-ination set up new i n f e c t i o n . When S. necrophorus reaches the p o r t a l c i r c u l a t i o n through abrasions, i t i s arrested In the l i v e r and abscesses are produced. These animals i n turn excrete more organisms, which i n f e c t or r e i n f e c t other animals, producing a new crop of abscesses. As the organism i s a s t r i c t anaerobe, the fresh dung of an animal with i n f e c t i o n i n the l i v e r w i l l be es p e c i a l l y dangerous. When there i s no crowding at the feed trough, the contamination i s very l i t t l e or Is completely eliminated. -57-( f ) Suggested Future Programme The conditions observed at the feed l o t s v i s i t e d during t h i s investigation support the hypothesis that crowding at the feed trough predisposes towards l i v e r abscesses. There-fore i t i s hoped to v e r i f y t h i s i n greater d e t a i l l a t e r by ( i ) C o l l e c t i o n at slaughter of b i l e and feces from animals with l i v e r abscesses, and i s o l a t i o n of the organism by rabbit inoculation and c u l t u r a l methods. ( i i ) C o l l e c t i o n at slaughter, f o r comparative study, of the same specimens from animals that have no l i v e r abscesses. ( i i i ) Determination of the l i n e a r space per animal at the feed trough. ( i v ) Comparing the figures for abscess incidence i n new and old feed l o t s . 2. Foot Rot (a) Introduction Necrobaclllosis of the foot i s a necrotizing i n f e c t i o n of the tissues immediately proximal to the coronary band, or of i n t e r d i g l t a l tissues, often complicated by a r t h r i t i s of the navicular Joint. The causal organism i s S. necrophorus, either alone or i n association with other organisms. The organ-ism enters through macerated tissue of the i n t e r d i g l t a l area. Wet weather, f i l t h and traumatic agents contribute to the mace-r a t i o n and i n j u r i e s predisposing to i n f e c t i o n . The moisture, mud and f i l t h contribute to longer s u r v i v a l of the organism by promoting more e f f e c t i v e anaerobic conditions. Contamination -56-of the s o i l may be by fresh feces or from denuded necrotic tissue of foot rot cases. (b) Symptoms Severe lameness and anorexia are the early symptoms. When necrosis has set i n , a c h a r a c t e r i s t i c f o u l smell i s present. In l e s s severe infe c t i o n s there may be only dermatitis. As the i n f e c t i o n progresses ,necrosis of the i n t e r d i g i t a l tissue and a r t h r i t i s of the c o f f i n j o i n t are seen. Secondary Involvement and complications bring about o s t e i t i s , l a m l n i t i s , inflammation of the connective tissue, and a r t h r i t i s of the pastern and fe t l o c k Joints. Laminitis may terminate In denudation of the horn of the hoof. Death i s due to toxaemia. Lesions are r a r e l y seen i n the v i s c e r a of animals dead of foot r o t . 3 . Calf Diphtheria (a) Introduction This i s one of the common forms of n e c r o b a c i l l o s i s occurring often as an acute f a t a l i n f e c t i o n of young calves. I t commences as a sporadic i n f e c t i o n and rapidl y assumes epizootic form. The f i r s t p u b l i c a t i o n on c a l f diphtheria i s that of Dammann,who i n 1876 published r e s u l t s of h i s investigations of t h i s disease. In the early period, many workers noted S.  necrophorus when dealing with t h i s disease, which resulted i n the organism aoquiring many synonyms i n d i c a t i n g i t s apparent r e l a t i o n s h i p with the disease. Here also i n f e c t i o n i s through breaks i n the buccal mucous membranes caused by teething or sharp objects. (b) Symptoms At f i r s t , the lesions i n the mouth or on the tongue resemble small scabs but l a t e r coalesce to form necrotic areas surrounded by a reddened zone. An offensive odour i s present. The necrosed tissue i s firm l y adherent to the under-l y i n g t issue. Later when ulceration has set i n , a thick wall of granulation tissue surrounds the ulcer. Necrosis may penet-rate into the deeper structures, causing loosening of the teeth and involvement of the bones, caseous nodules of the skin, and f a c i a l dermatitis. Prehension and d e g l u t i t i o n are affected, i n t e r f e r i n g with nursing. Death r e s u l t s from malnutrition and toxaemia. Very r a r e l y are lesions noticed i n the i n t e r n a l v i s c e r a . B. Horses (a) Introduction In horses the common i n f e c t i o n caused by S. necro- phorus i s gangrenous dermatitis. During the 1st World War th i s i n f e c t i o n among horses and mules of the U.S. Army ranked second i n importance only to influenza. During the l a s t s i x months of 1918, 4036 animals or 27.3% of the horses i n the army, were on sick l i s t , mostly due to S. necrophorus i n f e c t i o n . Nolecheck (1918) reported that at Hancock, Augusta, 20% of the horses became infected and &0% of the infected animals died. - 6 0 -Cesarl (1912) observed S. necrophorus i n horses associated with necrosis of the extremities, necrosis of the f i b r o c a r t i l a g e s of the phalanges '(.lavart cartllaglneux) g necrosis of the tendons "(javart tendineux)", necrosis of the hip ("gangrene de decubitus"), necrosis of the c e r v i c a l l i g a -ment ("mal de nuque", "mal d'encolure", "mal de garrot"), necrosis of the myocardium, and d i p h t h e r i t i c exudates of the i n t e s t i n e s . Quinlan et a l (1926) found that 25$ of the horses affected with gangrenous dermatitis died with lesions of gangrenous metastatic pneumonia. (b) Symptoms The lesions are seen on the coronary band and pastern, extending upwards to involve the f e t l o c k Joints and other portions of the body. Quittor and thrush i n horses are infeotions of the coronet and frog of the foot respectively, due to S. necrophorus. These infec t i o n s also show the charact-e r i s t i c necrotic tissue and malodorous discharge, and are promoted by insanitary housing conditions. C. Swine In swine, S. necrophorus causes b u l l nose ( i n f e c t -ious r h i n i t i s ) , i n f e c t i o n of the mouth and snout, causing swelling of the snout, deformity of the f a c i a l bones and death. This i s usually seen i n p i g l e t s about s i x weeks of age. In some piggeries i t i s of endemic character, i n f e c t i n g as many - 6 1 -as 25% of the annual p i g y i e l d . Nasal catarrh, mucopurulent nasal discharge and lacrimation are common c l i n i c a l symptoms. Post-mortem lesions include extensive necrosis of the mucosa of the nasal passages and f a c i a l sinuses involving the turbinate bones. D. Mink Mink often die suddenly and symptomless. At autopsy a thick greyish yellow mucopurulent exudate, contain-ing S. necrophorus i n association with other organisms, i s present i n the p l e u r a l cavity. The p a r i e t a l and v i s c e r a l pleura are t y p i c a l l y covered with a fibrinous necrotic exudate. Adhes-ions may wall o f f the thoracic cavity, and the lungs may be shrunken and a t e l e c t a t i c . E. Fowls In chickens, S. necrophorus causes avian diphtheria, characterized by scab formation on the head and comb si m i l a r to that of fowl pox. Necrosis i s circumscribed and i n f e c t i o n of the under-lying structures Is not extensive. This necrotic dry mass f a l l s o f f , revealing granulation t i s s u e . The f a t a l i t y rate i s n e g l i g i b l e . In a pigeon injected with v i r u l e n t culture, necrosis took place at the s i t e of inoculation. This necrotic mass slowly shrank and separated i t s e l f from the under-lying tis s u e and f e l l o f f . F. Rabbits (a) Introduction In rabbits, S. necrophorus causes a spreading -62-necroels at the s i t e of entry. Schmorl (1891) encountered i t i n a spontaneous enzootic among h i s laboratory animals, i n the form of necrotic ulcers, abscesses of the l i p s , skin and sub-cutaneous ti s s u e . Basset i n 1908, and Cameron and Williams i n 1926, also encountered the same condition. Current Observations Fifty-one out of 55 rabbits which received Inocul-ations of via b l e cultures of 3. necrophorus, i n dosage of 0.1 ml. or more, died of n e c r o b a c i l l o s i s within 2 to 55 days. In a l l , 11 f r e s h l y Isolated strains of S. necrophorus were used, each s t r a i n being tested on a variable number of (1-7) rabbits. Subcutaneous i n j e c t i o n of l i v e culture almost invariably pro-duced Induration of the s i t e within 24 hours. The swelling Increased i n size u n t i l about the f i f t h day, reaching 1-2 inches i n diameter (see plate l ) . By about the eighth day the swelling became ulcerated ( see plate 2). The ulcer increased i n area and depth and the sides were thickened. A f t e r l4 days the ulc e r was \ to 1 inch or more i n diameter and nearly £ inch deep (see plate 3)• In 4 rabbits the ulcer healed spontaneous-l y i n about 7 weeks. In one, i t broke out again a f t e r 2 weeks, and repeated t h i s entire process, twice during 6 months. Except for t h i s single external l e s i o n , a l l the 4 rabbits are a l i v e and healthy to date (6 months a f t e r i n j e c t i o n ) . One of the rabbits developed the syndrome seen by Schmorl, v i z . , spreading necrosis of the l i p s . A tenacious, cheesy, yellowish, malodorous nasal discharge blocked both the nasal o r i f i c e s , causing asphyxiation. The skin of the - 6 3 -face and areas around the n o s t r i l s had hard crusted eruptions resembling dermatitis. Post-mortem revealed fibrinous i n s p i s -sated pus f i l l i n g both sides of the median septum nasi, the nasal c a v i t i e s , and roots of the i n c i s o r s . Most of the rabbits developed abscesses of the l i v e r . Some had m i l i a r y abscesses of the l i v e r with fibrinous necrosis of the peritoneum, external surfaces of the inte s t i n e s , pleura, both sides of the diaphragm, and the pericardium (see plate 4 ) . A few showed a greenish-blue necrotic area of the contact surface of the v i s c e r a l aspect of the l i v e r , involving only the lower marginal borders. Five rabbits developed j o i n t i n f e c t i o n s of the legs which culminated i n t h e i r i n a b i l i t y to move. In 3 rabbits, the prominent l e s i o n was dis i n t e g r a t i o n of the wa l l of the stomach, r e s u l t i n g i n rupture on s l i g h t or no handling. The ruptured area showed only a thin outer membrane. Table 9 P r i n c i p a l Lesions i n 51 Rabbits That Died From ~"5 S. necrophorus Inoculation Lesions Observed Number Percentage 1. Emaciation 37 72.6 2. L i v e r lesions associated with other les i o n s . 32 62.S 3. L i v e r and lung lesions 23 45.0 4. Multiple necrotic lesions 16 31.8 Lung lesions only 53 10. 6. J o i n t i n f e c t i o n only 5 9.8 7. L i v e r lesions only 3 6. g. Brain i n f e c t i o n 1 2.0 -64-A l l oases of subcutaneous or intrap e r i t o n e a l i n j e c t -ions of l i v e cultures resulted i n l o c a l necrosis and abscess formation. Only 7 2 $ of the inoculated rabbits showed consider-able emaciation. In combination with other organs, the l i v e r i s the most commonly affected organ ( 6 2 . 2 $ incidence), followed by lungs ( 4 5 $ ) . In a r t i f i c i a l i n f e c t i o n s , multiple lesions are seen more often than infections of i n d i v i d u a l organs. J o i n t infections considered pathognomonic by Ce'sari ( 1 9 1 2 ) , occurred i n only 1 0 $ of t h i s series of rabbits. Brain involve-ment i s rare, occurring only once. There was no evidence that the pattern of lesions developed was dependent on the s t r a i n of S. necrophorus inocu-l a t e d . However, differences i n virulence were noted. For example, out of the 1 1 strains tested, one was nonlethal from the beginning; one became nonlethal after 5 subcultures; and another became les s v i r u l e n t a f t e r 3 months. G. Man ( a) Introduction There are few cases of human n e c r o b a c i l l o s i s on record. The e a r l i e s t report of the disease was that of Schmorl and h i s laboratory assistant, each of whom developed an abscess on one finger. Ellerman ( 1 9 0 5 ) i s o l a t e d S. necrophorus from the vulva of a nine-month-old infant dead of l a r y n g i t i s and pneu-monia. Stemen and Shaw (19J.0) reported an acute i n f e c t i o n in a government meat inspector, who accidentally scratched h i s hand on the teeth of a sheep, which had died of ulceration of -65-the l i p . Van Wering ( 1 9 2 3 ) described a case of necrobacll-l o s i s of the arm caused by the b i t e of a cow. Harris and Brown (1927) described an organism indistinguishable from 3. necrophorus as the cause of puerp-e r a l i n f e c t i o n . Wohlstein ( 1 9 2 9 ) , Van Gelder ( 1 9 3 0 ) , Cunning-ham ( 1 9 3 ° ) , and Shaw (l933)» reported various infections i n human beings; but i t was Dack and hi s associates who made comparative studies of the organisms i s o l a t e d from ulcerative c o l i t i s i n human beings and from bovine l i v e r abscesses. In general, strains i s o l a t e d from human lesions were less patho-genic than those i s o l a t e d from animal species. But there i s considerable v a r i a t i o n i n the pathogenicity f o r rabbits of st r a i n s of animal o r i g i n . Virulence does not appear to be associated with any d i f f e r e n t i a t i n g c u l t u r a l c h a r a c t e r i s t i c s . Judging from the va r i e t y of c l i n i c a l manifest-ations caused by t h i s organism and i t s wide geographical d i s t r i b u t i o n , human n e c r o b a c l l l o s i s i s probably more common than i s indicated by the reported eases. The d i f f i c u l t y of Is o l a t i n g anaerobics i n general, and S. necrophorus i n part-icular., i s probably responsible f o r the low recorded incidence. - 6 6 -CHAPTER IX. IMMUNOLOGY 1. Introduction Basset ( l 9 0 g ) was the f i r s t to attempt Immuniz-ation against 3. necrophorus. Using k i l l e d cultures he produced an antiserum i n a horse which protected guinea pigs against f a t a l doses of cultures given i n t r a p e r l t o n e a l l y . Bahr (1913) produced immunity i n goats and guinea pigs by c a r e f u l l y regul-ated doses of l i v e cultures given intravenously. They r e s i s t -ed l e t h a l doses given i n t r a p e r l t o n e a l l y and sera from them protected mice against f a t a l doses of culture. Ce'sari ( 1 9 2 1 and 1 9 2 3 ) , also using k i l l e d cultures, succeeded i n immunizing guinea pigs against the action of the endotoxin, but was not able to immunize rabbits against l i v i n g organisms given Intramuscularly. By using a mixture of culture and antiserum, he induced passive immunity i n rabbits. However, neither the antiserum nor the vaccine had any curative propertie Lignieres ( 1 9 2 3 ) was able to protect rabbits again-st natural i n f e c t i o n by using antiserum produced i n a horse, although they were not protected against challenge doses. This antiserum, nevertheless, showed curative properties i n lesions of avian diphtheria where S. necrophorus was a secondary invader Harnach ( 1 9 2 S ) inoculated rabbits with 0.2 cc. of whole culture intradermally, and a month l a t e r they r e s i s t e d normally f a t a l doses given subcutaneously. Beveridge (193*0 found that out of 12 rabbits i n -oculated, 4 developed a low l e v e l of Immunity, while 4 others - 6 7 -showed no immunity. In view of these various claims a series of experiments was conducted to f i n d out the degree of immun-i t y which could be induced i n rabbits and guinea pigs. 2. Present Project A. Object 1. To study the agglutinogenic power of the antigen. 2. To compare the agglutinogenic power i n rabbits and guinea pigs. 3 . To compare the influence of the route of i n j e c t -ion in the production of agglutinins. 4. To compare the degree of immunity produced i n rabbits and guinea pigs by vaccination. 5. To compare the potency of vaccines prepared by di f f e r e n t methods. 6 . To determine the s p e c i f i c i t y of agglutinins in sera. B. Materials and Methods Co l l e c t i o n of Materials from Slaughter Houses Portions of 66 abscessed l i v e r s were co l l e c t e d from animals at the time of slaughter at Vancouver, and one came from Kelowna. In 5 instances, i t was possible to obtain blood at the time of slaughter from abscessed animals. Blood was c o l l e c t -ed also from 3 ° animals free from abscesses.. S. necrophorus organisms were Isolated In pure culture from 55 of these abscess-es. The presence of t h i s organism was demonstrated i n 7 of the the remaining 12 specimens. Separation of Serum and Preservation After coagulation of the blood, the separated serum was siphoned off a s e p t i c a l l y into s t e r i l e serum v i a l s , and 10% phenol saline was added as a preservative . The sera were stored at 5 ° C Iso l a t i o n of Antigen The abscess was opened a s e p t i c a l l y , using a hot spatula for searing and a s t e r i l e s c a l p e l to cut through the capsule. Pus was transferred from the abscess to medium No. 1 and Robertson's medium f o r culture. A small quantity of pus was mixed with approximately 10 times i t s volume of nutrient broth and streaked on to 2 sets of agar plates containing 10$ defibrinated bovine blood. One set of plates was incubated ae r o b l c a l l y , and the other Incubated anaerobically as quickly as possible, using a C02 atmosphere i n most cases. (Sometimes pus was streaked on to B.A.P. without d i l u t i o n i n broth). A wet f i l m from the nutrient broth and pus mixture was examined under the microscope using a d a r k f i e l d condenser, and i f any organism other than S. necrophorus was present, the correspond-ing culture was discarded. The pus was stained by Gram's method and examined I f any organism other than S. necrophorus was detected, the corresponding culture was discarded. Wet films and Gram stain-ed s l i d e s of the cultures i n medium Nal and Robertson's medium were examined between 18-24 hours, and any culture that -69 -contained contaminants was discarded. The aerobic blood plates were checked f o r growth a f t e r 24 and 4g hours, and i f there was any growth, the corr-esponding culture was discarded. The growth on the plates was subjected to tests of m o t i l i t y , Gram-positivlty, gross morphol-ogy, c o l o n i a l c h a r a c t e r i s t i c s , and other tests to decide the family of organism present, and the plates were then discarded. The anaerobic blood agar plates were checked f o r growth a f t e r 4 g , 7 2 and 96 hours, and sometimes longer. To avoid death of the organisms from exposure, colonies were picked into medium No.l and Robertson's,medium immediately a f t e r open-ing the anaerobic jar, i f growth was present. The morphology, and the staining and c o l o n i a l c h a r a c t e r i s t i c s were studied only a f t e r the required number of t y p i c a l colonies had been picked. I f any contaminating organism was detected on the plate a l l the cultures corresponding to that i s o l a t e were discarded. The o r i g i n a l cultures In l i q u i d media were sub-eultured meanwhile every 24 hours, and I f any contaminating organism was detected a l l the cultures related to i t were d i s -carded. Other c h a r a c t e r i s t i c s such as production of HgS.,fetid odour, and l i q u e f a c t i o n of gelatin,could be read from medium No.l which contains the r e q u i s i t e indicators. NOTE: In the early stages of this Investigation, i s o l a -t i o n of the organism was carried out by plate cultures and shake cultures i n tubes. Later plate cultures were found un-necessary, f o r the following reasons: - 7 0 -(1) &5% of the bovine l i v e r abscesses contain S. necro- phorus i n pure culture. (2) I t i s not d i f f i c u l t to detect a contaminant because of the d i s t i n c t i v e morphological c h a r a c t e r i s t i c s , Gram's s t a i n reactions, and atmospheric require-ments of S. necrophorus. (3) Other workers have found l i q u i d medium sa t i s f a c t o r y . (Jensen,1954; Beveridge,1959; Prevot,1959). (4) Plates of fresh s o l i d medium have to be prepared every day or every other day fo r any success at Isolati o n . ( 5 ) On plates, growth i s slow to appear, and t h e i r use is therefore time-consuming. (6) S. necrophorus could be more e a s i l y , and Just as accurately i s o l a t e d i n the l i q u i d medium, with less labour and time. C. Preliminary Experiments A few preliminary experiments had to be conducted to decide the M.L.D., LD50 etc. f o r rabbits and mice. 1. Determination of M.L.D. for Rabbits The M.L.D. f o r rabbits was determined only f o r s t r a i n 60. This was done by i n j e c t i n g 1 rabbit with 0.05 ml., 2 rabbits with 0.1 ml., 3 rabbits with O.15, 4 rabbits with 0.2, and 2 rabbits with 0.3 ml., of a 24-hour culture. A l l those that received 0.2 and 0.3 ml. doses died i n 6-12 days. Those receiving lesser doses d i d not die. The injec t i o n s were - 7 1 -given intravenously. (For d e t a i l s see controls i n tables 1 5 an^' 17» Rabbits No. 55 and 56 injected with a d i f f e r e n t antigen are not Included). 2. Determination of L.D.^ Q For Mice The L.D.^Q of 6 strains of 3• necrophorus f o r mice was determined. When given i n doses of 0.05 ml. of a 24-hour culture i n t r a p e r l t o n e a l l y , 9 out of 12 mice died i n 6-20 days, regardless of the s t r a i n . When injected subcut-aneously none died. In doses of 0.025 ml., 1 out of 4- died on the seventh day. Therefore the L.D.50 i s somewhere between 0.025 and 0.05 ml. 3. Tests f o r Presence of Exotoxin To v e r i f y the claim of Cesari ( 1 9 1 2 ) and Orcutt ( 1 9 3 0 ) that 24-hour cultures contain exotoxins,supernatants of 24—hour cultures centrifuged at 5 ° 0 0 r.p.m. were Seltz-f l l t e r e d using a membrane f i l t e r pad. The f i l t r a t e s were injected i n t r a p e r l t o n e a l l y into 4- groups of 5 mice, each mouse rec e i v i n g a single dose. The f i r s t group were given 0.25 ml., and the other groups 0.5; 1 and 2 ml., respectively. None of them died. The supernatant of a 24-hour culture, centrifuged at 12000 r.p.m., f a i l e d to k i l l any mouse i n doses ranging from 0.1 to 2 ml. 4-. Tests f o r the Presence of Endotoxin When 24-hour cultures heated at 60°C for 1 hour were injected subcutaneously into mice and rabbits necrosis - 7 2 -of the underlying tissue occurred. Abscesses developed, which took up to 2 months to heal. This f i n d i n g suggests the presence of endotoxic substances. 5. To Test the E f f e c t of Oral Administration of Live Culture on Mice After withholding drinking water f o r 2 days, 4 mice were given 4 ounces of a 24-hour culture as drinking water. They consumed the culture i n 4g hours, and remained healthy u n t i l s a c r i f i c e d 2 months l a t e r . No lesions were seen on post-mortem. D. Immunization Procedure Experiment 1 Immunization of Rabbits and Guinea Pigs This experiment was designed primarily to produce high t i t r e s of agglutinins i n rabbits and guinea pigs. Groups of these animals were inoculated by d i f f e r e n t routes and with va r i o u s l y prepared antigens of the same s t r a i n of 3. necro- phorus . Altogether 12 rabbits and g guinea pigs were i n -oculated with vaccines as follows: Group 1 Four rabbits and 4 guinea pigs were inoculated subcutaneously with h e a t - k i l l e d vaccine. (For method of prepr aration see Appendix l . a . ) . Group 2 Four rabbits and 4 guinea pigs were inoculated subcutaneously with formalized vaccine. (For method of prep-aration see Appendix l . b . ) . -73-Group 3 Five rabbits were inoculated intravenously with a washed suspension of antigen. (For method of preparation see Appendix I.e.). A l l the animals received preparations derived from the same culture of one s t r a i n , No. 60, the i n i t i a l dose being 0.2 ml., and subsequent doses increasing by 0.2 ml. per inoculation, u n t i l 4- inoculations had been given at 3-&ay i n t e r v a l s . A f t e r a break of 3 weeks from the date of the fourth i n j e c t i o n , 2 doses of 1 ml. each were given at 3-day i n t e r v a l s . For the sake of c l a r i t y , the procedure i s tabula-ted below: Table 10 Details of Routes and Vaccines No. of No. of No. of Vaccine Route Dose i n ml. the Group Rabbits i n the G. Pigs in the Used 1st Ihjn 2nd 3hji. 3rd 3hjn 4th Ihjn 5th Iijn. bth 3hjn. Group Group 1 Heat-K i l l e d S.C. 0.2 0.4- 0.6 Q.g LO 10 2 4- Formal-ized S.C. n II n ti II II 3 5 Washed Suspen-sion I.V. II it it II II N Animals were bled Just before the f i r s t and f i f t h i n j e c t i o n s , and again 3 days aft e r the fourth and s i x t h i n j e c t -ions. The sera were tested f o r agglutinins and the r e s u l t s were as follows: -74 -Table 11. Agglutinin T l t r e s of Immunized Animals Group No. Rabbit No. Highest DI] Agglut! .ution of Serum i n which Lnation was seen. 1st Bleeding 2nd Bleeding 3 r d Bleeding 4 t h Bleeding 1 22 % 25 n i l n i l 1.10 1.20 1:2560 1:2560 1:5120 1:10,240 1 :320 1 :320 1 :640 1:1280 1:1280 1:640 1 :320 1:2560 2 26 27 28 29 1:40 1:80 1:10 1:20 l:5 1 2 0 1:640 1:256O 1:5120 l i e d 1:80 1 :320 1:1280 1 :320 1:1280 1:2560 3 30 3 1 32 ll 1:20 1:10 n i l n i l n i l l :iq240 1:5120 1:1Q240 1:5120 l:5 1 2 0 1:1280 1:1280 1:1280 1 :640 1:1280 1:5120 1:10,240 1:2560 1:256O 1:10,240 1 Guinea Pig No. 1 2 I n i l n i l n i l n i l 1:80 1 :320 1:640 1 :640 1:40 1 :160 1:80 1 :640 1:40 1:80 1:80 1 :320 2 i 7 2 n i l n i l n i l n i l 1 :160 1:160 1:40 Died 1:80 1:20 1:80 1:80 1:20 1:40 Summary of Result The table shows that agglutinins are produced to considerable t l t r e s , though i n d i v i d u a l animals respond to d i f f e r e n t degrees; eg. the highest d i l u t i o n of the serum from rabbit No. 27 with agglutinable antigen was only 640, while that of the sera of rabbits 25,30 and 32 was 10240, 3. necrophorus evidently is feebly agglutinogenic for guinea pigs. In rabbits, the intravenous route yielded a higher response than the subcutaneous route. Very l i t t l e or no d i f f -erence was apparent in the agglutinogenic properties of heat-k i l l e d and formalized vaccines. The agglutination reactions were noted as 4 . + + + , +++, ++> +» and the titre taken is the highest dilution in which a + was seen. It is noteworthy that a prolonged taper-ing off effect in the agglutination reaction was seldom seen. In some instances the pattern was + + , + + + , ++++> . . . . . ++++» + + + , + + , + . But often i t would end more abruptly as + + + + , + + , 0. The same abrupt tapering effect was noted by Orcutt (1930) in her studies. The + + + + represented very few large floccules floating in absolutely clear supernatant f lu id and + was in fine granules with clear supernatant, and + + + and + + were intermediates. The control suspension showed a uniform slight opalescence without any sedimentation. For preliminary experiments relating to determ-ination of optimal conditions for agglutination reactions see Appendices 6 - 9 . To measure the titres of cross agglutinins in the sera of these rabbits and guinea pigs, their sera were subjected to agglutination reactions using three heterologous strains^numbers 63,65 and 66 and compared with the reaction against i ts homologous antigen No. 60, Results are tabulated in Table 12, on the next page. -76-Agglutination Reaction of Sera of Rabbits and Guinea Pigs  Immunized with Str a i n 60 And Bled Three Days A f t e r Four Inoculations Table 12 Group No. Serum of Rabbit No. Antigen Number 60 63 65 66 1 22 9 25 2560 2560 5120 10,240 •10,240 5,120 5120 1280 1280 2560 2560 1280 2560 2 26 27 28 29 5,120 640 2,560 5,120 5120 1280 5120 10,240 1280 640 640 5120 1280 320 1280 5120 3 30 31 32 P 10,240 5,120 10,240 5,120 5,120 20,480 20,480 5,120 20,480 20,480 5120 2560 1280 5120 5120 10,240 5,120 1,280 10,240 5,120 Guinea P i g No. 1 1 2 I 80 320 64o 64o 80 640 640 640 40 320 640 320 SO 320 320 640 2 I 7 160 160 4o 320 80 40 160 80 160 320 80 80 The r e s u l t s indicate that the sera possess heterol-ogous agglutinins i n considerable degrees. The sera of rabbit numbers 3°,31,33, and 34 agglutinated the heterologous antigen s t r a i n No. 63 i n higher t i t e r s than i t s homologous antigen -77-s t r a i n No. 60. The sera of c a t t l e with and without abscesses were also tested f o r cross agglutination reactions and the res u l t s are tabulated below. Table 13 Agglutination Reaction of Sera from Cattle with Hepatic Abscess Antigen Number \ Serum No. 60 63 65 66 60 % 66 2560 I28O 1230 1280 1280 5120 640 1280 1280 1280 1280 T2~m 320 640 1280 10,240 Table 14 Agglutination Reaction of Sera of Cattle Free from Abscess Antigen Number Serum No, ~TTo~ 111 112 1 X ? 114 116 nI 118 119 120 121 122 123 124 12 12 127 128 129 130 "6T 320 80 160 160 80 320 2560 320 640 160 80 640 80 320 40 160 160 40 80 40 160 80 160 320 80 320 5120 320 1280 160 SO 1280 320 1280 320 320 1280 40 160 80 s: 320 40 20 20 40 0 20 320 40 160 320 80 2560 320 1280 160 160 2560 80 160 80 bb 160 80 40 80 40 0 40 640 160 640 640 160 5120 320 2560 320 320 2560 40 80 30 The culture of 3. necrophorus tested were those i s o l a t e d from hepatic abscesses of the same animal from which serum was also c o l l e c t e d . Sera No. 110 to 130 were from c a t t l e found to be free from abscesses. The 4 s t r a i n s tested possessed a large measure of common agglutinogens. Serum No. 60, 63, and 66 showed a high t i t r e against the respective homologous s t r a i n s . Serum 65 however, showed equally high t l t r e s with the homologous and 2 of the heterologous s t r a i n s . The differences i n t i t r e s shown are accountable by variables. But in some instances the range of t i t r e s i s too great to be accounted f o r , because the maximum range of t i t r e s shown by any serum against the 4 antigens tested had approximately an g to 1 r a t i o . In several instances higher t i t r e s were shown by a given serum against a heterologous antigen than against i t s homologous antigen. The same phenomenon so f a r unexplained was observed by Beveridge (1934) and Feldman et a l (1936). Even i n the case of sera of abscess free animals, there was marked differences i n t l t r e s . The serum of the same cow reacted d i f f e r e n t l y to the d i f f e r e n t antigens, eg., sera No. 117 and 127 gave a range from 1:320 with antigen 65 up to 51g0 with anltigen 63. Serum 127 gave a t i t r e of only l : l 6 o with antigen 60 while with 65 and 66 i t rose up to 1:2560. Other sera, eg., 112, 12g, and 130 had given generally low t i t r e s ranging from 1:20 to l:gO against a l l the 4 antigens. - 79 -The Pattern of Adsorption In the course of these agglutination experiments, the microscopic appearances of the agglutinated organisms were occasionally investigated. A peculiar pattern of coherence was noted. In cases where coarse f l o c c u l e s appeared, i t was found that the focus of coherence was at the terminal or sub-terminal points of the c e l l , and that groups of four c e l l s were often seen agglutinated together i n a rectangular pattern. Large masses of agglutinated organisms presented a network of such patterns with plenty of free space within the rectangles. (See Plate 6 ) . Y forms, V forms, and ra d i a t i n g forms are also regularly seen. P a i r s of organisms were r a r e l y seen l y i n g i n p a r a l l e l s , while palisade formation was never noted. Many long filaments may be noticed with short rods attached only at the ends (See Plate 7) and occasionally one migjit see the f i s h bone arrangement, which again proves that the points of coherence are situated terminally to convey the message. Type of Agglutination Reaction Noticed Two types of reactions were noticed. One was the f l o c c u l a r type with white f l u f f y masses f l o a t i n g i n a c r y s t a l c l e a r medium. This i s e s p e c i a l l y noticeable i n the tubes as soon as these are removed from the water bath. On standing at room temperature, i t sediments, r i s i n g when s l i g h t l y shaken. Vigorous shaking w i l l break the f l u f f s into smaller p a r t i c l e s , leaving the medium s t i l l c l e a r . - go -Floccular agglutination i s seen when the antigen i s prepared from young cultures i n which long filaments pre-dominate. Low centrifugation speeds and c a r e f u l handling during reconstitution, are also necessary to prevent the "break-down of filaments. The other type seen i s the granular type. This reaction i s seen when older cultures with predominantly coccoid forms and short rods are used i n the preparation of antigen. The reaction i s seen as f i n e granules i n a clear medium. Occasionally a compact, rod-like mass, similar to that seen i n the b r u c e l l o s i s agglutination reaction i s seen. A hand lens i s very useful for reading t h i s type of reaction. Experiment No. 2 - Challenge of Immunized Animals. To compare the degree of immunity produced by S. necrophora8 i n rabbits and guinea pigs, and to compare the potency of d i f f e r e n t preparations of vaccine, the rabbits and guinea pigs i n groups 1 to 3 were challenged intravenously with a 24-hour l i v e culture of s t r a i n No. 60 which was used f o r t h e i r immunization. From experience with 10 strains previously tested for l e t h a l i t y , i t was presumed that the MLD would be 0.1 ml. Accordingly, the rabbits i n each group were sub-divided (as shown i n Table l 6 ) and given 0 . 3 , 1.5, and 3.0 ml. ( 3 , 15, and 30 MLD) of culture intravenously. The actual MLD was determined simultaneously by i n j e c t i n g 9 rabbits with various amounts of a culture of s t r a i n No. 60, as shown in Table 15. - 31 -Table 15 Determination of M.L.D. of Strain No. 60 for Rabbits No. of Dose Day of Post-mortem Group No. Rabbit in ml. death aft e r lesions challenge Controls 42 0.05 43 0.1 44 0.1 45 0.15 46 0.15 47 0.15 43 0.2 9 Joint lesions 49 0.2 10 Pneumonia and necrosis of lungs 50 0.3 6 Multiple necrotic l e s i o n s . The r e s u l t s indicated that the M.L.D. was 0.2 ml. and hence the challenge doses were i n f a c t equivalent to 1 ,^ 7^ and 15 M.L.D. The re s u l t s of the whole challenge exper-iment are given on the next page. - 8 2 -Table l 6 The Results of Challenge (1st) of Immunized Animals j 1 Group No. No. of Rabbit Dose i n ml. Day of death a f t e r challenge Post-mortem lesions 1 22 3.0 11 Multiple v i s c e r a l lesions 23 3.0 7 Pneumonia and Liver necrosis 24 0.3 - — 25 0.3 - — 2 27 3.0 9 Purulent urine S. necrophorus i s o l a t e d from blood 28 0.3 - — 29 1.5 - — 3 30 0.3 - — 31 0.3 - — 32 0.3 - — 33 1.5 - — 3* 1.5 - — 1 G-. Pigs 1 2 3 4 o.5 0.5 0.5 0.5 3© Small necrotic focus on l i v e r 2 5 6 7 0.5 0.5 0.5 — - S3 -The r e s u l t s indicate that vaccinated rabbits were able to r e s i s t 1.5 ml. M.L.D.). In order to obtain some ind i c a t i o n of the degree of immunity i n the survivors of the challenge dose, they were again divided into groups and re-challenged a f t e r IS days with 5, 15, and 25 M.L.D. of either the homologous or a heterologous antigen. The heterologous antigen selected was a culture of s t r a i n No. 6 3 , whose a g g l u t i n a b i l i t y was s i m i l a r to that of the homologous antigen. Five rabbits were used to determine the M.L.D. for s t r a i n Number 60 and 6 3 , and the results are tabulated below. Table 17 Determination of M.L.D. of Strain Number 60 and 63 f o r Rabbits Group No. Rabbit No. Antigen No. Dose i n ml.. Days of Survival Post-mortem lesions Control 52 60 0 . 3 A r t h r i t i s shoulder 53 60 0 . 2 7 P e r i t o n i t i s and multiple lesions 54 6(3 0 .2 15 L i v e r abscess 55 63 0 . 2 12 Pneumonia 56 63 0 . 3 6 Necrosis of the endocardium and p e r i c a r d i t i s . The results indicate that the M.L.D. of s t r a i n 60 i s 0 . 2 ml., and that of 63 i s roughly of the same order. / - ,64 -Table 18 The Results of Second Challenge of Immunized Animals Group No. Rabbit No. Antigen No. Dose i n ml. Dose i n Terms of M.L.D. Day of Death Post-mortem lesio n s . 1 24 60 1.0 5 26 a r t h r i t i s 25 60 3.0 15 6 a r t h r i t i s 2 28 29 60 63 5.0 1.0 25 5 2 14 P e r i t o n i t i s and l i v e r necrosis Abscess i n abdominal muscles. 3 30 60 3-0 15 10 Multiple lesions 60 5.0 2 5 3 Necrotic f o c i i n l i v e r 32 63 3.0 15 7 Pneumonia 33 63 3.0 15 5 Pneumonia 34 60 5.0 25 9 Abscess of face with involve-ment of brain. Summary of Results of Test f o r Immunity 1. A l l the vaccinated animals showed immunity against 7 i M.L.D, but not against 15 M.L.D. Rabbit No's 30,31, 33 and 34 were not able to r e s i s t 15 M.L.D. i n spite of the fact that t h e i r sera showed a high t l t r e up to 1 i n 20,000. This i s not at a l l su r p r i s i n g as i t i s commonly seen that agglutinins are not an essential component of the defence mechanism. 2:. The method of preparation of the vaccine, whether h e a t - k i l l e d or formalized, has no influence on the development of immunity. - S5 -Only one of the guinea pigs developed a minor necrotic l e s i o n , i n sp i t e of the low t i t e r of agglutinins dev-eloped by a l l 7 vaccinated guinea pigs. . Vaccinated rabbits which had survived the f i r s t c h a l l -enge, and were rechallenged with a heterologous c u l t -ure, showed s i m i l a r degrees of resistance to others s i m i l a r l y challenged with the homologous culture. The f a i l u r e of rabbits 24 and 29 to survive the second challenge dose of only 5 M.L.D., and the death of a l l rabbits surviving the f i r s t challenge, when rechallenged with 15 or 25 M.L.D., suggests that the f i r s t challenge d i d not s i g n i f i c a n t l y r a i s e the degree of Immunity. - 36 -CHAPTER X. DISCUSSION The characters of strains i s o l a t e d conform c l o s e l y to those described by e a r l i e r workers. S. necrophorus s t r a i n number 696 D of the Basteur I n s t i t u t e , P a r i s , obtained f o r comparative study from Doctor A.R. Prevot, resembled morphologically, c u l t u r a l l y and biochemically those i s o l a t e d during this i n v e s t i g a t i o n . However Doctor Prevot 1s s t r a i n was l e s s pathogenic than most other strains i s o l a t e d here, In that the former caused extensive abscesses but not death. Sl i g h t differences i n gas production, patho-g e n i c i t y , v i a b i l i t y i n laboratory media, tending to form fusiform swellings and a g g l u t l n a b l l i t y exist between str a i n s . Yet on the whole may be considered that these strains constitute a f a i r l y homologous group. Intravenous inoculations of l i v e cultures produced i n some rabbits Joint infections considered path-ognomonic by Cesarl; but more often lung lesions resulted. There i s no organ of p r e d i l e c t i o n not even the l i v e r , for n e c r o b a c i l l o s i s . The f a c t that S. necrophorus infections of that organ are so common, only indicates the frequency of bacteremia and inevitable access to the l i v e r through the p o r t a l c i r c u l a t i o n . Inoculation of dead cultures produces agglutinins i n the system, the intravenous route being more agglutinogenic than the sub-cutaneous; but immunity i s low i r r e s p e c t i v e of the route of administration. Individual and species variations e x i s t i n agglutinogenssis, the rabbit being a more agglutinogenic species than the guinea pig. - 87 -Though les s pathogenic to the l a t t e r species, 3. necrophorus seriou s l y i n t e r f e r e s with or arrests the growth of young animals. The apparently normal c a t t l e without apparent abscess presumably show a high t i t e r of agglutinins for one of the f i v e following reasons a. They had been infected, but the dose was not large enough to cause abscess formation, although s u f f i c i e n t to stimulate antibody production. b. They had been infected and developed abscesses, but because the host developed antibodies rapidly, the i n f e c t i o n was aborted. c. They had been infected recently, and at the time of testing were a c t u a l l y incubating the i n f e c t i o n . The i n f e c t i o n had not so f a r resulted i n abscess formation, while the body responded promptly and produced agglutlns. d. They never had been inf e c t e d with 3. necrophorus, but with another organism having a s i m i l a r a n t i -genic structure. The l a s t of these alternatives offers the lea s t p l a u s i b l e explanation, since the "normal" ca t t l e concerned came from herds which included some cows with abscesses. - gg -CHAPTER XI. SUMMARY 1. S. necrophorus was i s o l a t e d i n pure culture from 55 out of 67 "bovine hepatic abscesses. 2. Eleven strains out of these were i s o l a t e d and studied. 3 . Various media were t r i e d f o r s u i t a b i l i t y of growth-promoting and growth-sustaining q u a l i t i e s . Their merits and de-merits are discussed. 4. The use of f e r r i c ammonium c i t r a t e i n 2.5$ strength i n culture medium Is advocated. 5. The Importance of an adequately heavy inoculum i s emphasized. 6. The optimum temperature of incubation of cultures, the pH of the medium, and the pH changes due to growth, have been studied and stated. 7. The c u l t u r a l , biochemical, morphological and staining c h a r a c t e r i s t i c s have been studied and noted. g. By 4-hourly observations, i t was found that the period of peak growth, provided other conditions are optimum, i s between 16 and 24 hours. 9 . The process of l y o p h l l i z a t i o n Is described. 10. The optimum age of culture f o r harvesting and the method of preparing antigen suspensions i s explained. 11. Thirteen rabbits and 7 guinea pigs were inoculated by d i f f e r e n t routes with k i l l e d cultures of S. necrophorus. Agglutinins i n t h e i r sera, before, during and a f t e r inoculation were measured. 12. The immunity developed i n experimental animals thus inoculated was estimated i n terms of MUD of l i v e cultures of 3.necrophorus administered intravenously. 13. The pathogenicity of the organism fo r rabbits i s described and i l l u s t r a t e d by 3 colored plates. 14. The post-mortem lesions of rabbits, dead of m i l i a r y necrosis, are described and i l l u s t r a t e d by a colored p l a t e . 15. The MLD of s t r a i n 60 f o r rabbits and the LD 50 of 6 s t r a i n s for mice have been determined. 16. The cultures were tested for exotoxins and endo-toxins. No exotoxin was detected. Heat-killed cultures produced necrosis of the area of inoculation on subcutaneous i n j e c t i o n into rabbits. 17. Sera from c a t t l e with abscesses were c o l l e c t e d at the time of slaughter, and tested f o r agglutinins, using homologous and heterologous strains as antigens. 16. The sera from apparently normal cattle free from abscesses were c o l l e c t e d and tested f o r agglutinins. They were found to give s u r p r i s i n g l y high t i t r e s . The possible reasons for such findings are l i s t e d . 19. Sorensen's buffer solution with 0.3 per cent formalin at pH "J.k as a medium for suspension of antigen for agglutination test was found to be good because of the clear contrast i t presents between agglutinated and nonagglutinated tubes and i t s keeping q u a l i t y . - 90 -20. The optimum temperature and period of incubation i n the agglutination reaction was found to be 5 6 0 C for 8 hours. 21. The F l o c c u l a r and granular types of agglutinations were observed. The f l o c c u l a r type was given by the organism i n the filamentous phase and the granular type when the phase was cocco-bacillary. 22. The point of coherence between agglutinated organisms i s terminal or subterminal. This i s i l l u s t r a t e d by 2 photomicrographs. 23. Literature on the subject has been exhaustively studied and sparingly quoted. O r i g i n a l i t y Is claimed for items 4, 5, 8, 9, 10, 19, 20, 21 and 22. - 91 -CHAPTER XII. APPENDICES 1. Preparation of Vaccine (a) Heat K i l l e d The supernatant of a pure 24-hour culture i n Robertson 1s medium was siphoned off without any s o l i d p a r t i c l e s , into a s t e r i l e f l a s k and heated i n a water bath at 6o°G f o r one hour. The concentration of organisms per ml was adjusted to 1C-7 using nutrient broth, and the suspension was stored at 4°C. (h) Formalized. The supernatant of a pure 24-hour culture i n Robertson's medium was syphoned o f f as described above, and formalin added drop by drop while shaking, to make a f i n a l concentration of 0.5 per cent. I t was then incubated at 3~[ 0Q for one hour and t i t r a t e d for the number of organisms per ml. as above, and stored at 4°C. (c) Washed Suspension. The supernatant of a pure 24-hour culture i n Robertson's medium was syphoned off and sub-jected to the same treatment as for Heat K i l l e d Vaccine. It was then centrifuged at 5000 r.p.m. for 20 minutes. As soon as the centrifuge stopped, the supernatant was slowly poured out to the l a s t drop without disturbing the sediment. - 92 -Ten ml. of phenol saline was then added to the sediment i n each tube, reconstituted, and re-centrifuged as before. A f t e r pouring out the supernatant, s u f f i c i e n t phenol saline was added to make a f i n a l concentration of organisms per ml. This was checked by the percentage of l i g h t transmlttance (L.T.) using a Beckmann Colorimeter and was found to be 15. I t was then stored at 2. (a) Preparation of Antigen f o r Agglutination. The supernatant of a pure 2*r-hour culture i n Robertson's medium was syphoned o f f without any s o l i d p a r t i c l e and centrifuged at 5°00 r.p.m. for 20 minutes. The sediment was mixed with 10 ml. of Sorensen'e buffer solution contain-ing 0.3 per cent formalin at pH 7.^ and recentrifuged. The sediment, after the second centrifugation, i s mixed with the same diluent and the concentration of the organism adjusted to SO per cent l i g h t transmlttance. Formalin not only k i l l s and preserves the organism, but also prevents contamination r e s u l t i n g from frequent handling. (The meat p a r t i c l e s i n Robertson's meat medium contain many organisms which are l o s t i f only the supernatant of the culture i s aspirated. This l o s s can be avoided by washing the meat p a r t i c l e s three times with Sorensen's buffer solution and adding the washings to the supernatant. During the process of washing by shaking, fine s o l i d p a r t i c l e s became suspended and are aspirated. I t i s d i f f i c u l t to get r i d of these by centrifugation. - 93 -Under such circumstances, the L.T. of the f i n a l suspension i s adjusted to about 60, and the suspension l e f t undisturbed f o r 24 to 43 hours, by which time a l l s o l i d p a r t i c l e s sediment. The supernatant i s syphoned off c a r e f u l l y without disturbing the sediment, and the L.T. adjusted to 80). (b) Agglutination Technique. Agglutination reactions were set up i n the-usual agglutination tubes with 2 ml. of antigen i n the f i r s t tube, and 1 ml. of the antigen i n the subsequent tubes of the s e r i e s . F i r s t t i t r a t i o n s were done i n d i l u t i o n s of 1:10 to 1:320 using s e r i a l double d i l u t i o n s . I f there was agglutination i n any of the l a s t two tubes, the t i t r a t i o n was repeated i n 10 tubes, s t a r t i n g with 1:40 d i l u t i o n s and reaching by s e r i a l double d i l u t i o n s to 1:20430. 3. (a) Preparation of Phenol Saline for Preservation of Serum. Phenol saline was prepared by adding 5 per cent phenol and 0.85 per cent Na.Cl. to d i s t i l l e d water and s t e r i l i z i n g In the autoclave. The f i n a l concentration when added to the serum i n 10 per cent strength, would be 0.5 per cent phenol and 0.085 per cent s a l i n e . (b) Preparation of Phenol Saline for Use as Diluent f o r Washed Suspension.  Phenol saline was prepared by adding 0.5 ml. phenol and 0.1 gm. NaCl to 100 ml. of d i s t i l l e d water and s t e r i l i z i n g . (c) Preparations of Sorensen's Buffer Solution a * 7^  PH Sorensen's buffer solution i s prepared by d i s s o l v i n g 37.74 gms of Na2HP04 i n 4 l i t r e s of d i s t i l l e d - 94 -water, and 9.02 gms. of KH^POj^ i n 95° ml. of d i s t i l l e d water. The two solutions are mixed and autoclaved, a f t e r which 15 ml. of formalin i s added. 4. Method of Calculating Number of Organisms per ml. These calculations were made by mixing 0.01 ml. of the suspension to be t i t r a t e d with one ml. of d i s t i l l e d water, and spreading evenly 0.01 ml. of the d i l u t e d suspension over a glass s l i d e having a marked area of 1.69 sq.cm. (13 mm. to a side of a square.) The number of organisms per f i e l d was counted i n 10 f i e l d s under a magnification of 450, using a dark f i e l d condenser, and calculated by applying the following formula: N ml. - A N F. X H X n|j where N ml. = Number of organisms per ml. A N F = Average No. of organisms per f i e l d . as = area of spread (1.69 cm.) af • area of focus (area of the f i e l d under focus) Q, = quantity used as was found to be very close to a* 100 and i s always constant. The quantity used, v i z . 0.0001 ml. Is also always constant. as „ ml = . „ .„ 1 - _ ~6 a f X -q- 100 X 1 10 10000 Therefore N ml. = Average Number of organism per f i e l d X 1 0 6 . NOTE: There i s a s l i g h t error in accepting | | as 100 because the area of the f i e l d under focus i s 1.6742>3 sq. mm. and not exactly I .69 sq. mm. - 95 -There could also be v a r i a t i o n i n the d i s t r i b u t i o n of the organism i n the area i n which i t i s spread, with the r e s u l t that the average of 10 f i e l d s may not be the true average. The v a r i a t i o n i n size of the i n d i v i d u a l organism also i s great. By working with organisms grown under known common conditions of medium, inoculum, age, etc., the v a r i a t i o n i n sis&e could be narrowed down considerably, but not eliminated completely. However, the formula i l l u s t r a t e d w i l l give s u f f i c i e n t accuracy f o r p r a c t i c a l purposes. 5. Method of L y o p h l l l z a t l o n A 24-hour culture of 3. necrophorus i n Robertson's medium with 1% glucose was aspirated a s e p t i c a l l y into tubes i n 30 ml. quantities and centrifuged at 5°°0 r.p.m. for 20 minutes. The supernatant was poured o f f as soon as the cent-r i f u g e stopped, and skim milk at the rate of 3 m-L' P^1" tube was added and reconstituted into a thick emulsion. One ml. aliquots of t h i s was transferred to l y o p h i l i z i n g tubes and freeze-dried i n Edwards' c e n t r i f u g a l Freeze Dryer, Model L . 5 and sealed. The cultures were viable when tested a f t e r 3 months. 6. Determination of Time and Speed of Centrifugation. Various t r i a l s were made to assess the optimum time and speed so that complete sedimentation without mutilation of the organism could be obtained, and the following are the findings for a 20-minute period. - 96 -1. There Is no sedimentation of the organism at less than 1000 r.p.m. 2. Some sedimentation takes place at 2000 r.p.m. 3. Almost a l l the organisms sediment at 5000 r.p.m. 4. At 5000 r.p.m. the sediment i s l i g h t l y packed and w i l l become dispersed i n the super-natant i f i t i s not poured off without much delay. 5. At 5000 r.p.m. there i s no mutilation of the organism, and filaments as long as 5° or more microns are seen to survive two cent-rifugations at t h i s speed. 6. When the speed of the centrifuge i s more than 7500 r.p.m., the sediment i s t i g h t l y packed and there Is mutilation of some organisms. 7. At speeds over 11,000 r.p.m. there i s complete mutilation of a l l long filaments and only small rods are seen i n t a c t . In the l i g h t of these findings 5000 r.p.m. for 20 minutes was adopted with the precaution that the supernatant should be poured o f f as soon as the centrifuge has come to a stop. ( S e r v a l l Type KSA - 3(4) Superepeed Centrifuge was used for a l l centrlfugations). 7. Standardization of the Antigen Concentrations by Light Transmlttance. 1. As too heavy or too l i g h t concentrations make the reading of the reaction more d i f f i c u l t , - 97 -i t was necessary to determine the optimum concentration which would f a c i l i t a t e the read-ing. This was assessed by a series of t r i a l s which showed £0% l i g h t transmittanoe to be optimal. g. Determination of Temperature and Period of Incubation. To decide the optimum temperature and period of incubation, a series of experiments were conducted wherein tubes were Incubated at 22, 37» and 56°C for varying periods. I t was found that agglutination takes place at 22°C i n 72 hours,eat.37 C i n 24 hours and at 56 C i n g hours. The easiest f l o c c u l e s to read seemed to develop a f t e r g hours at 56 C. An equally complete reaction could be obtained by a double phase incubation, v i z . , at 56 C for 2 hours and at 22 C. f o r 12 hours. Agglutinated tubes can be held for many days without much a l t e r a t i o n i n the reaction. In t h i s study the routine procedure adopted was to incubate tubes at 56 C for g hours. 9• Other Factors A f f e c t i n g Agglutination. Saline was found to bring about spontaneous agglutination i n concentrations above 0.5$. Cultures of antigen grown i n Robertson's medium gave less trouble i n this respect than those i n medium No. 1. 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From l e f t to ri g h t -(a) perforated ulcerations of the large intestines. (b) a mass of necrosis, marked by the points of the forceps. (c) necrotic membrane covering the l i v e r and (d) m i l i a r y necrosis of the diaphragm (e) lungs. - 1 1 2 -PLATE 5 Eighteen hour culture showing rods and filaments, (a) fusiform swelling (b) old filaments showing metachromatic granules. (c) note absence of metachrom-a t i c granules in young filaments (x 95°- enlarged 13.75 times i n p r i n t i n g ) . PLATE 6 Smear prepared from agglutination tube. Note the network of rectangles ( l ) x 95° - enlarged 13*75 times i n p r i n t i n g . A - 1 1 3 -PLATS 7 Agglutination reaction showing point attachment at rig h t angles (x 950 - enlarged 13-75 times in printing.) PLATE S Agglutination reaction i n tubes - showing f l o c c u l a r type of agglutination.. Note the sedimented floccules i n tubes 1-4 and Suspended floccules i n 5-7-No agglutination i s visable In tubes 8-10, and control tube. 

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