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A study of the mechanisms of haemolytica immunity to Pasteu. rel. la infection. I HELEN BARBARA EVANS A thesis for submitted University the degree of 1979 ZG dý8N10ý of Edinburgh Doctor of Philosophy DECLARATION The work project concerned diseases of findings were at the sheep, and Research work presented and where conjoint was played in design of the results. thesis was in the a large±: of respiratory with Institute. experiments part some of collaboration this of of consequently in obtained Moredun this investigations with the tation in reported the experimental my colleagues Nevertheless, thesis carried out necessary, a was were experiments most and the by of myself full role interpre- W O1 C-/aAAs HELEN October EVANS 1979 CONTENTS Pa qe No DECLARATION Summary 1- Acknowledgements 4 Abbreviations GENERAL in used text INTRODUCTION Characterisation of The distribution healthy amongst Diseases of P. haemolytica Septicaemia 2. Pneumonia P. of P. sheep sheep 1. for Requirements P. haemolytica haemolytica haemolytica associated Experimental in sheep Septicaemia 2. Pneumonia Models of laboratory 7- 38 10- 17 18- 19 20- 26 vaccine for study against of 27 infection with of P. pasteurellosis haemolytic animals GENERAL MATERIALS AND METHODS Bacteriological techniques Strains of P. haemolytica Strain of Escherichia Enumeration Laboratory 6 with a suitable and methods reproduction 1. 5- types efficacy vaccine 3 of animal Mice Inoculations 1. Subcutaneous 2. Intraperitoneal 3. Intranasal 28- 31 32- 38 39- 56 39- 41 42- 43 in coli bacteria techniques Vaccines page No 44- 48 49- 56 57- 88 58- 59 Antigens 1. Preparation of P. 2. Preparation P. haemolytica of heat-killed Preparation of Absr rption hydroxide of Emulsification oil mineral of Bovine vaccine Techniques collaborators for sheep P. in antibodies lambs with P13 virus and bacteriology and Quantitative P.. haemolytica enzyme-linked P. lungs mouse sera Pathology 1- of haemolytica Challenge of SPF P. haemolytica Chapter to responses examination test with antigen transformation Lymphocyte P. haemolytica antigens IHA aluminium onto absorbed by employed Histological SSE vaccines antigens albumin haemotica to estimation of antibody in sheep sera by a micro(ELISA) immunosorbent assay Experimental infection of mice with haemolytica Introduction Materials and Experiment varying with 1- Methods Intranasal numbers of 2Viable Experiment in the lungs of mice Experiment thiopentone intranasal Experiment live and infection P. haemolytica of mice 60-62 counts of P. haemo]_ytica infection after 3- Comparison of halothane for sodium as anaesthetics infection 4Comparison of IN ii_ection heat-killed P. haemolytica 63- 64 65- 67 68- 70 and with Page No 71- 72 73- 74 75- 77 8The effect Experiment of of vaccination P. haemolytica type Al vaccine mice with in the liver of organisms upon the growth following IP challenge P. haemolyti. ca with type Al 3 weeks later 78- 80 Discussion 81- 88 5- Experiment procedure; challenge Intraperitoneal challenge for preparation of inocula of mice and determination of Experiment type Al in 6- Growth of the livers and P. IP LD50 haemolytica of spleens normal mice Experiment SC routes type tica 7prior Al Vaccination to challenge 2Mechanisms Chapter in sheep ytica of of immunity mice by IP and P. haemoly with to P. haemol89-108 Introduction Materials and Methods ]Growth Experiment of in the presence in vitro and serum P. of haemolyti ca. lung wash cells 2Experiment The roles of antibody immunity (CMS) in combined mediated P13 virus of SPF lambs with and P. type Al 91- 92 93- 95 and cellinfection haemolytica 97-103 104-108 Discussion 3Non-specific Chapter P. haemolytica resistance to 109-129 Introduction Materials and Methods 1The effect Experiment given of Carovax 24 hours to mice 48 hours, and 6 hours to challenge prior upon the LD of P. haemol. y50 tica 110-111 112 Page treatment to prior of growth 2Experiment Effect of vaccine intervals time mice at different P. haemolytica on with challenge in the liver bacteria Effect of dilution of challenge with 4Experiment dual components Effect of treatment on of the vaccine 5- Experiment B188 with of Carovax P. haemolytica 116-117 P. with challenge of 113-115 3Experiment on the outcome type Al of No Al type haemolytica Determination indiviwith the outcome LD5 of of 'treated . E. co" untreated mice and in mice 24 hours before challenge vaccine in 118-119 120-121 6Effect with Experiment of treatment 24 hours before Carovax with challenge B188 upon the growth E. coli of the organism in the livers of mice 122-123 Discussion 124-129 4Chapter and challenge into investigations Further P. haemolytica of mice with vaccination 130-1153 Introduction Materials and 1Experiment of P. haemolytica mice and and challenge 135 A2, Al, types with A6 136-138 A9 Experiment procedures 3upon P. haemolytica Effect of challenge type 4-16 Experiments types different of by the model gated P. haemolytica with Experiment P. of LD Q values Zack for C57 Determination strains 2Vaccination a of mice with (types Al, A2 and A6) P. haemo3ytica_ Experiment trivalent vaccine 132-134 Methods haemolytica challenge Discussion 17 - 139-142 A2 Cross-protection P. haemolytica, of IP challenge P. between investiof mice 143-144 Vaccination type with vaccination various of mice with A9 SSE haemolytica of mice with and HKO, and typt- A9 145-147 148-153 Page Chapter 5- for tica detection from sera the in Development IHA of and to of antibodies vaccinated mice No tests ELISA haemoly- P. 154-121 Introduction Materials and 1- Experiment for dilutions 157-159 Methods Determination mouse of in sera IHA end-paint tests homologous and heterologous using SSE extracts P. haemolytica 160 2Preliminary titration Experiment of P. haemolytica type Al SSE in the presence to of immune mouse serum and titrations dilutions determine optimal of conjugate 3- Experiment Titrations of sera types of P. haemolytica antigens dilution conjugate using at 1/200 comparable achieve at absorbance 4Experiment of cross-reaction P. haemolytica and 161-163 SSE Al-A12 to 400 nm 164-165 degree Determination of the between the types in the. ELISA test of 166-167 168-171 Discussion DISCUSSION CONCLUDING Mechanisms of 172-190 immunity to P. in haemolytica 174-1.81 sheep Models of P. haemolytica infection in mice 182-190 191-212 Bibliography Appendix 1 213-214 Appendix 2 215-217 Publications arising from this thesis. 218 1 SUMMARY to Immunity using was studied in infection haemolytica P. of models haemolytica Pasteurella both lambs and mice. Immunisation .vaccine of specific a sodium containing P. haemolytica type Al P. haemolytica type Al, type parainfluenza of responses increased lambs transfer this although by haemolytica alveolar from by from to P. vaccinated The passive repeated did säure vaccine, upon failed serum leucocytes Serum effect with transformation produced challenge. with Lymphocyte serum, not protect lambs vaccinated in haemolytica facilitate vitro, phagocytosis in macrophages a of infection upon challenge. the with challenge against after with a bacteriostatic had P. blood lambs from recipients 3 virus. markedly of vaccination extract superimposed hyperimmune of salicylate protected peripheral lambs pathogen-free of vitro. w Two models were caused cleared because similar haemolytica Intranasal investigated. llytica P. of lesions from in the'lung lesions infection the by were in infection lung of mice 3 days 48 hours produced later. after bj the were mice with P. haemoBacteria infection, introduction and of 2 heat-killed haemolytica, P. this model was investigated not further. P. haemolytica gastric vaccination of tica sodium containing types various The to type Al, commercial 24 hours or 14 days no such before detection vaccinated of mice. vaccine was immunosorbent There to P. was of between mice the P. haemolytica that was given with. P. was used Carovax. vaccine, challenge, indicating challenge antibodies challenge cross-protection with protection An enzyme-linked haemolytica challenge before coli, the Vaccines against protected Pasteurella challenge When non-specific. haemoly-" P. observed. the Escherichia also The of P. of in spleen. investigated. little intraperitoneal against protected T10 of 12 or and extracts was -and was liver extracts and strains, investigate 7 days All strains When given and A8, model liver the intraperitoneally mice various salicylate homologous with the A6, Al, the with in growth upon to in multiplied mucin effect introduced vaccine type Al, protection 1 hour, was 48 hours, haemolytica observed. assay haemolytica considerable was in developed the sera cross-reaction for of between sera extracts of from the mice different immunised types with of P. sodium salicylate haemolytica. 4 Acknowledgements am deeply I Dr. G. H. out this grateful to for their Lawson K. project, thanks My sincere Sharp, M. Dr. J. for many and for are also Dr. N. Burrells for advising aspects, Dr. B Rushton on Mr. pathology, house, Mr. pbs, Mr. B. would librarians reading. Research Council. A. like also typing and Mr. at this L. to McVittie Mr. A. Suthe-rland to the Moredun manuscript Throughout Studentship this awarded Mr. I was the and the advice animal of photograI assistance. Evans project by help of and Miss A. technical many for C. statistics, Skeoch J': Donachie Mr. preparation Institute, and and technical Miss W. to staff on for for thank the advice Inglis Mr. and W. Angus and for Aitken, D. tests Mr K. through- encouragement. am endebted me on ELISA C. I. Dr. Gilmour I and and advice due J. M. McLauchlan Easter and their and supervision excellent discussions. useful P. W. Wells Dr. G. for Miss A. Sclater, for Armstrong help supported Agricultural C. with by proof. a Research - 5 Abbreviations AM = alveolar BA = bovine C. = control CFA = complete cfu = colony CMI = cell-mediated DW = distilled E= in used text macrophage albumin Freund's forming adjuvant unit immunity water absorbance ELISA = gelatin-Hanks = Hank's immunosorbent enzyme-linked 0.1% balanced salt solution, assay containing gelatin HBSS = Hank's H. E. = haematoxylin HKO = heat-killed HuRBC = human IFA = incomplete IHA indirect = IN = intranasal IP intraperitoneal = IT = intratracheal balanced solution salt and eosin organisms blood red cells Freundes adjuvant haemagglutination intravenous IV = MIF = macrophage MV missing migrätion value inhibition factor 6 broth NB = nutrient ND = not done n. t. = not treated density OD optical PBS phosphate-buffered saline PBS/Tween phosphate-buffered saline Tween P13 = containing 20 type parainfluenza PVP polyvinyl RBC red SC subcutaneous SE standard error SED. standard error 3 pyrrolidone blood S. I. = stimulation SPF = specific SSE = sodium V = vaccinate cells of difference index pathogen salicylate free extract of means 0.05% INTRODUCTION GENERAL is Pneumonia of sheep necropsied it cause, considerable in least its bacteria, lungs of the the organism is 1954). with a variety also of studied predominantly basis of important not cattle in of and Particular associated the certain types with is to is c and have organism pneumon'. a and Europe been into subdivided and and or America biochemical the may sheep, pneumonia P. haemolytica of the a septicaem North species from conditions restricted both with characteristics which with at Pasteurella clinical other associated of is similar of is The characteristics features. which isolated may be frequency greatest most (Carter, the some of associated fever" on is commonly "transit types disease is P. haemolytica extensively the from stages, the organism to terminal from disease. industry the losses pneumonia, Organisms sheep, may not report The the isolated the consequence. haemölytica. be that likely 1977). economic and with total the is in quoted reflect accurately this figures the proportion (Veterinary Report, II Analysis a high Kingdom United the Diagnosis. Investigation Although in in lesion important an ý antigenic are other types 8 with in septicaemia reviewed the points salient 1967; be will. been extensively 1973) Thompson, a later at considered only and point Vaccines still aimed in available that inadequate, vaccines this at are in that many the insufficient The general unsatisfactory pasteurellosis use immunising nature of vaccine field the by supported trials vaccine of This current against is possibility the of powers protect haemolytica disease. of uptake or are P. nevertheless outbreaks to latter disease. of but the either or prevention country*, implicated indicates the (Pyke, 1966). Development hampered to in introduction. this is (Carter, recently has This sheep. in reproduce the of past the *Carovax, Wellcome Ovivac-P, Pasticidin, Middlesex, England. haemolytica P. by the disease absence either Foundation Hoechst has vaccines in Ltd., of a suitable sheep or Beckenham, Pharmaceuticals, in been system laboratory England; Hounslow, is 9 This animals. development These not immunisation, satisfactory mechanisms has of a full involved aspects will in also only hindered but it progress has of protection against be discusscý prevented also understanding in towards immune the the this organism. introduction. 10 Characterisation taxon, The (1932) Cross and in disease In of a study by the isolated of reactions. non-motile, gram-negative in diameter haemolytic. 48 hours Cultures and organisms, and pneumonic (Spray, bacilli (1932) "typical" biochemical were and and "typical" was similar The "atypical" were which organisms, and to an cocco- Newsom haemolytic II as týicum ovise "atypical" Groups to isolated organisms, cattle, were referred were gram-negative and mm These of sheep short, 3-5 indole-negative. III and translucent, a study from (1921). organisms named isolated group of were In a groups Pasteurella 1923). defined flat Similar lambs bovine cocco-bacilli. non. -haemolytic, bovisepticus. Bacillus Jones and II 1921). of incubation, were Group indole-positive from were the of cases organisms bipolar agar after I with (Jones, morphology, Group serological on blood three colonial Newsom associated earlier from distinguished criteria Colonies years by description first the eleven bacteria Jones pneumonia an organism although appeared introduced was designate to cattle, had organism haemolytica P. haemoiytica P. of and group. and III group Cross The of resembled 11 Group Jones' I and (The "typical" Pasteurella was has group it is but does not produce reactions of species as classified and and attacks certain were adopted are (Cowan P. facultatively fermentation, 1974). Steel, and anaerobic. by sugars haemolytica diverse, are and identification criteria-for by Biberstein, listed non- a gram-negative, as aerobic gas of this, These (1960). been since defined oxidase-positive Cultural view is which rod, is haemolytica. Pasteurella multocida). P. haemolytica motile designated in the and Knight Gills here, of in diminishing order The of of importance. 1. Haemolysis on blood haemolysis strain, considerably varies and directly P. haemolytica distinguished, is sometimes underneath haemolysis agar. is the a useful and as from However, colonies. by which feature latter strain detectable only P. multocida the extent can is be non-haemolytic. to ,2 i 2. Absence of uniform characteristic in indole to contrast P. a baemolytica, P. of is This production. multecida, is which indole-positive. 3. Growth on grows well P. 4. In McConkey's does multocida Failure to is"oxidase-positive, it from most the milk. haemolytica P. characteristics, feature a useful of blue methylene these wbereas agar, not. reduce to addition McConkey's on haemolytica P. agar. distinguishes which Enterobacteriaceae, which are oxidase- negative. Satisfactory bacteria are fermentation often fermentation difficult media inconclusive strains biotypes, designated be associated is achieve, poor, of with P. as 1973). haemolytica A and T. growth pneumonia, and on many obtained Smith from Biotype these with and results (Thompson, classified to to reactions (1959 sheep A strains biotype are B) into two appeared T strains 13 with lambs. of septicaemia characterised further fermentation reactions, to colonies grey small arabinose biotype while brownish but not lost blood but in to penicillin of methods sensitivities larger by 10 days, colonies trehalose with 2 days, within A strains Biotype broth ageing formed fermented and trehalose not morphology, A strains agar, 10 days. within and Biotype sheep later were colonial curves formed rapidly viability of fermented and arabinose more sensitive and cultures were- than and tetracycline T strains. biotype A variety haemolytica P. subdivide that established from on T strains centres basis 1961). 7 days, within biotypes growth (Smith, antibiotics the on These whereas revealed one 1950), Godbille, Bosworth and haemolytica concerned mainly or Glover, and in three Biberstein, Gills to the an species with by distinct Cross, 1932), haemolytica two (Florent and (Montgomerie, means and the P. 1930), serotypes 1938), studies antigenically and to attempts Early Newsom Edington, reactions. subdivide used was 1921; (Tweed been serologically. (Jones, da P. multoci. studies P. have of Knight basis of agglutination (1960) somatic attempted antigens 6 14 by direct and arthritis types II) and ion be basis the on to expected of septicaemia, of nasopharynx by this into these and These method. a wide cover fell a two spectrum groups main fermentat- additional tests. P. haemolytica the subdivide into species (IHA) developed was antigen (1960). blood (bovine cells haemolytica in raised reaction by and rabbits their with the of species other and into between IAA tests types have This were IRA defined sharply also red antisera strains. eleven Knight from antigens with as bovine of agglutination cross-reactions A variety Gills soluble selected this using sensitisation against divided and RBC) An serotypes. test, to successfully used Biberstein, involved test The been several haemagglutination indirect types, has which heat-stable a soluble, possesses component, capsular P. the examined haemolytica, P. of were would which strains, from from derived cattle pneumonia, and mastitis, individuals, healthy and sheep bacterial autoclaved using conditions, clinical of variety (I from Strains cells. of tests, agglutination minimal. been developed 4 15 to 51 classify of fever these strains (HuRBC) cells dilutions test rabbits. All strains were the found red that if the study, the of sera haemolytica, the found be to from by instead used were agglutination treatment after cells, IHÄ test presence was modified dilute from the distinct cells red red against were clearer in suspended titres P. pig 0 human in test cases rabbit normal was employed immunised sheep rather than to Gills and Knight to with a type strains. unknown The been typed, were of strain was this This to polysaccharide raised totally and study. obtained antigen, determine it IHA test widely of be Biberstein, As used. became prepared antigen would it from agglutinated in Guinea isolated type onto sera and In has then were this attempted soluble immune in plasma. T'e adsorbed homogeneous HuRBC, known was (1966). patterns Canada. specific multocida Cameron haemoiytica of strains with in (1956) Carter P. of which serologically of haemolytica. strains shipping from of P. subdivide agglutinated apparent from larger that a particular by 2 or numbers of 3 antisera strains a batch occasionally strain (1960) of P. were of haemolytica (Biberstein, 1965). 16 In this some cases original different were due the typing to trays (Shreeve, strains P. divisions of and classified tation tests degree of representing as either (Biberstein correlation In to (1960) biotype biotype was A by Group found and is which to by to was the the basis was fermen- of A reasonable two methods s biotype and Group IT. (1959 A Knight An as classified Group of serotype Gills in two a selection each the T Biberstein, this, Smith'. included biotype 1962). Biberstein, 2, study. system relate between T corresponded by previously this A and T on Gills, general, I. of serotype Smith, A or and was tubes suggested haemolytica, correspond exception to P. biotype with 1972). To confirm classification. and later these Perspex a microtitre seemed species modified in biotype the of was Wasserman Thompson, and interfere substituted (1960). Knight of strains to that not discovered adapted haemolytica the who the was discovery of technique the cross-reactions were did in types of demonstrated was (1966) for Biberstein Smith's Gills was there which The serotype test the Later, antigens, capacity A 12th used. It Thompson 1 ml a mixture others procedure. and of in to types. minor Biberstein of but culture, between due was II of " B) 17 Biberstein, Gills 6,7,8,9,11 biotype A and biotypes A and homology between between a biotype of used to 1975). of vision one, and regarding haemolytica P. the to The P. the biotypes available haemolytica into feel biotypes as 1965). This brevity strains Al" and has not of "Type A and evidence been etc. these separate which (Thompson and that there is groups grounds species (Smith will for be the be Mould, subdia valid for and sake referred each broadly the are so of could groups two resolved, P. haemolytica T3 ", two T was hybridi- which suggests that homology the proteins patterns into of Electrophoresis constituent species degree (30-50%) 1968). gave some workers the the between interspecies Francis, of but T strain for obtained gels subdivide corresponded A strains, to T. a high showed a biotype and polyacrylamide serotype 'type often biotype a^-. d homologies nucleic haemolytica A and 1,2,5, belong to ?.U to and biotype (Biberstein sations in P. two that than of T of Serotypes now considered 3,4 serotypes investigation An less 12 are and (1960). Knight and Thal, of to as 18 distribution The haemolytica P. of types healthy amongst sheep The of study of P. the upper has respiratory tract 1970). Thompson, that the same types with the possible Type A2 whereas 5,8 and some healthy types 9 were well the tonsils being Sixty-four of P. contrast, of 95 of type 100 T 100 (Gilmour, adult survey and only showed the that the as Type was isolated 65% of and from these these 1974). Fraser, these were proportion A of nasopharynx sheep, 6% of such Britain. Great swabs 1966). USA, the Thompson not was Britain, haemolytica P. showed Britain, which Great in nasopharyngeal haemolytica A 12 month in T strains In flocks Thompson, in represented type A12, in rare Shreeve Biberstein, and predominant apparently sheep. isolates yielded were healthy USA and Great type flocks frequently normal of the of healthy apparently 1966; (Biberstein USA outnumbered strains from strains in exception in'the identified of A survey occur in the assisted isolated been Thompson, and has types haemolytica P. of haemolytica (Biberstein sheep different of epidemiology sheep. from and recognition sheep of of type T. sheep 19 in haemolytica P. carrying late from to no and weather the flocks in the this nasal of periods known is survey pattern P. of of to interesting types There of carriers that, although the haemolytica enzootic and flock. healthy, remained carriage Shreeve incidence between It changes. flock and markedly different the of month, si:, 1p1e relationship was with Prevalence month increased (Biberstein, spring 1970). Thompson, varied and autumn intranasally two peak correlated pneumonia in the region. lambs Young increasing number and Thompson, detected strains at birth, contact the lamb and with with of their after as birth. from adult sheep P. haemolytica. may grew typable In lambs likely appears ewes they The. first recovered it with types 1970). 48 hours were reared older facilitate (Shreeve were colonies few contrast, from removed that an acquired intimate their and colonisation typable dams prolonged of 20 Diseases 1. of sheep P. haemolytica with Septicaemia Fatal septicaemia often and preceded not by described Stamp, quantitative no has Post-mortem bronchi the capillaries and and interlobular seldom the from A; is an important and in heart blood, can lambs with Thompson, haemorr- kidneys under type 1966). and three A strains In on the on large the are found in cultured of is the be liver, associated and beneath and in ia blood'splashing neck lung, septicaemia and is of lungs the inflammation there the oe with enlarged are trachea distension However, nodes the of fluid, lung, te exudative haemolytica Biberstein frothy of Lymph mucosa, predominantly 1960 Although septicaemic congestion septa. P. Generalised include venules peritoneum, the of sudden first was disease this is (1955). study made, death which symptoms, blood-stained, with abomasal pleura. in Thomlinson and been Haemorrhagic visceral other Watt pneumonic. hagic. by findings and alveoli lambs, death. of cause of epidemiological pasteurellosis is associated parietal numbers spleen. months of (Smith, contrast, age 21 100% of due type to Thompson has yet suddenly to been offered as be more of the winter The first all common in days. The start of deaths The invasion have of Stamp, support Watt this. to be and perhaps Thomlinson In several and may be be amongst The as cases found where of the in several best the to primary site has been blood invader, no a flock 20% and, sheep It a secondary (1955) as known. may enter for turnips. after sheep identified. not and with days as high predispose is or stop which P. haemolytica tract, respiratory factors October rape a few all appear pastures ground on in would coincides period within to P. haemolytica that suggested yet tend occurs September, folding rate mortality should but year, sporadic are explanation syndrome low to occur Deaths organism This the condition. condition 1978). often this during (Gilmour, sheep convincing the of hoggs sheep surprisingly, of times high deaths moved. why found normally are are age 1966). Thompson, no The from or being to pathogenic. (Gilmour, November movement and 1974), of months healthy of Fraser, at and tonsils and sheep, of ages the become three over haemolytica P. of in commensals lambs (Biberstein T strains T strains Type as in septicaemias evidence pneumonic this of the via but to lesions 22 were it present, bacterial to 2. was the these pulmonary were secondary vessels. Pneumonia the One of losis of Bosworth sheep earliest be to Glover, and disease the means is constantly is not a specific of and of of outbreaks rarely phase acute usually of present, pericardium, disease, with in be extending but which the term haemolytica. becoming Other pleurisy usually during 1978). ill severely sheep such as may exhibit coughing such. acute In the are and pericarditis gelatinous the to applied pasteurellosis or to country term the 10% (Gilmour, into this P. with a greenish so community, to dying incidence, low Mortality exceeds the of involvement, discharge. oculonasal by strict disease. respiratory mild (Montgomerie, which pneumonic respiratory obvious Wales pneumonia", come sheep in pasteurel- referred associated with pneumonic authors However, acute of These a given has sheep suddenly, signs in one. pneumonia Outbreaks was 1938). present pneumonia" with described a pneumonia "enzootic begin outbreaks as "enzootic definition the in thrombi that considered anterior exudate over mediastinum. 23 large Also, fibrin or enlarged and areas. Such arc purple straw-coloured oedematous with the cases bright red oedematous and large with are areas fluid pleural hyperacute In observed. are clots lungs of volumes solid, haemorrhag-c. and septa are are distended in present feature this of areas surround associated 1961), of tissue The has proved in type lesions found the lung be inter- capillaries Spindle- cells. are and "oat cells" a characteristic numbers of these cells alveoli. necrotic in the called Large is A strains are The blood red fluid with and nuclei, pasteurellosis production to lung containing with which and pneumonia.. Pneumonic are oedema, basophilic the filled the of necrosis cocco-bacilli. fluid with show spaces widened'by with cells shaped alveolar gram-negative abundant lobular are the and alveoli lungs the Histologically, of numbers almost P. exclusively (Smith, haemolytica exceeding 107 per gram lesions. of difficult.. experimental Dungal pneumonia in (1931) produced sheep typi- 24 cal in pneumonia inoculation animal at reproduce by of P. haemolytica P. haemolytica type peritoneal cavities resembling the (Smith, transmitting mice, produced been along (1957). Downey of in cultured the infections pulmonary pneumonic Intrabronchial P. by numbers of heat-killed of numbers large had of to in achieved which form failed suspension A, acute 1964). of was material success was three infected chorioallantoic culture One all (1957) of Partial inoculation Intrabronchial in pneumonia inoculation routes. (IT) haemolytica. P. Salisbury infected with pneumonia with by intratracheal of while necropsy. pneumonia a variety cultures symptoms, showed by sheep 9 hour of demonstrated three pasteurellosis inoculation haemolytica of did similar produce not lesions. The strains commensals factors fully in which the predispose with more cause'disease sheep but one Parainfluenza susceptible to to type invasion Experimental*pneumor. specific free pathogen (SPF) there disease. lambs. (P13) 3 of the ic has that as must These is suggestion P. haemolytica. reside normally Therefore, nasopharynx. characterised, infection sheep which are be not prior virus may render lungs with pasteurellosis been-successfully in 25 by produced Gilmour, to Thompson and the. to support important an that other part in factors in The whether P. haemolytica to haemolytica invade, were most useful, a single agent its many an as would if exclusion factors which effective of be and documented, or is host and be would of easier important. little for place disease the could the against an environmental factor by management. However, disease than vaccination attempting be would vaccine by to sheep predispose pasteurella the many allows and by vaccination or, control viruses factor Control achieved by pasteurellosis, the there agent, important an P. haemolytica pasteurellosis vaccine. infecting of recognised play well a single compromises be more effectively is be (1968). to sheep is lends might etiology respiratory tract of only there to question factor if susceptible Loosli one were it which by involved, However, exist reviewed If primary the respiratory predispose infection in between finding This P13 virus may well sheep the 1978). factor Synergism been a P. that (Sharp, haemolytica P. of Rushton, theory rendering bacteria aerosol predisposing infection. factors an infection a P13 virus of pasteurellosis. pneumonic has establishment exposure to prior the to to exclude 26 a multiplicity of other factors. 27* for Requirements and a suitable but able, P. these are of due assess, to have attempts it tently successful. the organism, of experience P. personal not it made vaccine cal are nature to Due and have Such to to evaluation features of use or the for their disease species determination in these to Many pasteurell- been Downey, consis- distribution in haemolytica these has have sheep occurs. 1957; sheep IHA suggest disease. the ubiquitous purposes. P types difficult this antibodies the to Salisbury, of by experimental difficult the of pneumonic that majority detectable susceptible 1931; to it, of reproduce recently vast for evidence preparations communication). unsuitable are only yet 1978). These types of no avail- (Gilmour, number different made are widespread. the the haemolytica as (Dungal, is and is is sporadic been 1957), value vaccine experimentally osis questionable between the efficacy pasteurellosis there and trials vaccine against a limited cross-protection Field of haemolytica P. against pasteurellosis only haemolytica, that of use, contain vaccines study vaccines their Despite for methods Commercial vaccine of prior serum to (Gilmour, test be may therefore Laboratory animals infection for either of animals. which the has initial immunologi- 28 reproduction of pasteurellosis in reproduction of P. haemolytica septicaemia Experimental 1. Septicaemia Successful in sheep lambs was derived cultures (Stamp, Watt injection of P. 109.9 colony given IV in blood both with from of number very large less than death. factors disease. 24 hours, only one tends This are number involved As of to in septicaemia of adult in and adult dead produce type survived had haemol positive tics was the is septicaemia hitherto follows resul- sheep which which development often haemolytica adult sheep lambs. young and bacteria that Adult A). However, sheep, suggest the P. sheep. to from organisms whereas required especially P. of Lambs numbers 12 hours killed lambs B). three 1960 which old 1960 smaller within (cfu) units organisms the doses (IP) into A cultures death of Intraperitoneal (Smith, 6 month at cultures recovered in to (Smith, death challenge type injection septicaemia of 1955). peritonitis forming to cases haemolytica susceptible not were clinical resulted (IV) intravenous the Thomlinson, and fibrinous acute ted from lambs old week by achieved of is slightly only cause would undefined the a change field of T 29 was pasture, it sudden change For this by dietary thought diet, of reason, implying 2. haemolytica was in sheep susceptible to challenge were with no more is alone not respon- communication). that of Most large to consistent reproduction 1931; promising most method intrabronchial involved haemolytica type numbers of P. similar to acute pneumonic sheep have reared haemolytica, P. in of across which the lambs newborn indicating immunoglobulin with (Dungal, success The which sheep A. pasteure- produced. antibodies low, 1957). conventionally levels in pneumonia limited only (1964) infections were reproduce Downey, Smith of Pulmonary llosis to achieved 1957; inoculation very factor. induced was acidosis personal attempts Salisbury, bulin to the Pneumonia Early P. acidosis sheep that by caused a predisposing prior These (Gilmour, sible of manipulation, disease, acidosis, may be a state P. haemolytica. the that that the prior to is little placenta the may affect disease. there circulating ' Serum are sucking (Smith, or immunoglo- no Wells, generally transfer Burrells 4 of 30 and 1976). Dawson, colostrum received in challenge exposed to an aerosol has P13 virus after and transient develop severe animals (Sharp, illness were either agent P13 virus and success of (1978) Rushton method was attributed of have The time encountered interval may also be SPF lambs with haemolytica P13 virus 1971). Thompson Gilmour, reared less Thompson, and use with and severe Three both received infections combined the of 1978). Rushton, inoculated Sharp, consistently proportion lambs between important. 1969). a high Angus a produces in and P. respiratory ovine less 1975). Angus, and which to developed pneumonic lambs Shreeve, SPF lambs P. haemolytica and were the Al in hysterectomy-derived not challenge than (Biberstein, consistent ion. Thompson haemolytica P. in Previous alone. type Stevenson, and disease severe ten with and P13 virus lung'lesions more of not reproducible inoculation, (Hore Gilmour, Five Smith associated clinical and agents may well both give natural Thompson, experimental with to haemolytica from been disease mild inoculated Lambs P. of (Gilmour, pasteurellosis The experiments. have which expected 4.ndistinguishable pneumonia The be might results disease lanbs Therefore, and SPF lambs. of in before isolation inoculat- infection days elapsed and 31 between Shreeve, Biberstein, interval the This (Gilmour, Martin, of a series this challenge antigen was type with of A6, the and HKO of P. haemolytica haemolytica described investigate sheep type the which Later this mechanisms pneumonia. (HKO) model of tested. elsewhere one type P. of against of immunity but This for the thesis, this infection to types in Al challenge demonstrated. useful Al haemolytica experiment, extracts in the when of protection extremely sodium haemolytica in A2 was vaccines. in strain P. In demonstrated homologous A2 no proved fully more contained type has L. vaccine 1979). was was protection the of protection either for Wells, haemolytica preparation some where infection in extract A2 afforded of are the A heat-killed A6. another, and an Gilmour, preparations and discussed Briefly, with P, of be used immunogenicity the will against of whereas Sharp, of vaccine Thompson (SSE) . method method (1971) successfully experiments, thesis. the haemolytica Sharp, experiments Thompson in been extracts salicylate or P. of and the (1978). has method investigation in 7 days was in challenge Angus and Rushton Thompson These bacterial and viral model study of experiments was used 'P. haemolytica to 32 Models infection of laboratory Experiments with the number and costly A model seasonal. laboratory low and cost infection of available the small are is and throughout in of relative Unfortunately, year. has haemolytica P. a numbers of greater the with limited infection of animals SPF lambs, advantages availability animals experimental particular haemolytica have would ready in animals P. of animal animals sheep, of in haemolytica P. with proved difficult. Many by various routes. rabbits pigeons, Hughes, 1935; Glover, 1938). for pathogenic are to have workers and direct large inoculum In contrast, exceptionally guinea Beveridge, mice, and action unless the of (Smith, the virulent is very lethal the relatively large numbers endotoxin Lovell Bosworth also effects ania1als hens, 1931; Montgomerie, 2 haemolytica small dogs, cats, (Dungal, pigs 1937.; infect to vain include -These administered, the in tried and nonof may well present and in organisms be due such a 1958). related for organism, mice. P. is multocida When introduced by 33 various fatal P. the routes, subcutaneous with two doses IV suspension were later by IV, showed that manner by the immune of P. Another member is isolated and mouse show Rogers the high, no of clinical and outbreaks pulmonary by studies The this extremely the rate treatments various given Pasteurella, agent tract. mice can However, in (Collins following and mouse P. is Pasteurella of mice carry the organism Wetmore, pneumotropica frequently colonies and is incidence Its (Hoag, disease pathogen, pneumonia protection infectious but 7 days challenge routes. genus 1962). Meier, c' of multocida mediated. respiratory is P. eliminates mice when Mice achieved studied a natural from signs host the colonies potential in of also humorally a variety organisms immunisation in of route. Partial was been multocida by pneumotropica, in has 1-2 against Passive was LD50 (SC) SC routes. protection challenged often protected 1976). be causes a heat-killed of challenge which growth and and organism virulent of IP Woolcock, and fully aerogenic against to the and The shown or-by vaccinated rapidly 1973). 5A was strain IP IV, given grows (Collins, septicaemia multocida organism is implicated (Brennan, a 34 Fritz by and aerosol P. with organi. an from 1973). 1965; Flynn, In and prevented the synergistic the investigation mice interval virus This model effect. of maximum was and thus in useful proved bacterial and viral It virus infection, has effect infection 6 days. Sendai between synergism been virus was with Dick, and had the between subsequent the of which. virus, immunisation previous completely (Jakab killing P. pneumotropica with challenge that the in Sendai with when achieved shown delayed was challenged eliminated 7 hours intrapulmonary infected previously Mice alone within contrast, P. pneumotropica being pneumotropica lungs the 1950). Jawetz, infections. (1958) Smith inoculation of in resulted The haemolytica organisms were and the conjunction 100% mortality, of the'organisms susceptibility inoculation with of TV infection provided hydrolysate to confined Brain Intracerebral mice. casein of trauma the a suitable solution and brain individual induced P. death. and mice in mice solution, starch-saline with of methods successful in in usually considerably. intracerebral two multiplication bacteria meninges, varied P. with challenge developed haemolytica challenge by in gave dose was used. 35 To by given are This is mice route, needed. mucin gastric in intraperitoneal the organisms if infection establish be can incorporated haemolytica with P. very large numbers considerably into of reduced the inoculum enhance the (Smith, 1958). is It which of organisms by Olitzki, 1948). pneumococcus and mice. Bacillus Jourdonais IP into bacteria, than upon Wolf, greater injected 1953). organism, It depletes serum of properdin, important role in the to infection. showed that properdin ability However, there activity of mucins. was in act suggested vitro investigation correlation host, of by rather not that of of towards if equal, pathway and-the the which resistance direct viscosity role been a protein an no the of the injected albicans probably has (Nungester, rats upon into IP protective alternative lowering thus into Candida which are III injected Although factors other (Reviewed type IP a physically importance. activation, include and virulence non-pathogenic pneumonia 1936) has the or Examples (Scherr, mice can weakly anthracis and the are mucins Klebsiella evidently mucin that accepted mucin. plays an complement the animal DeWitt between infection-promoting (1958) anti- 36 The infection IP incorporated into valuable occur of the from sheep tica and test". then (Gilmour, personal protected against 1959 A). investigate in immunity from bacterins were subsequently gous strains. suggested roles determining the of of of with resulting the specificity somatic antigens of only which of the immunity secondarily (Smith, this model antigens vaccinated homologous play the be also somatic capsular pattern antigens the of used were haemolytica challenged capsular and and sera haemoly- P. can (1965) Mice somatic with immunisation active capsular P. the determined Mice P. haemolytica. strains IP mice be Thompson and The that by into against can mice with some measure sera challenge combinations various the that developed communication). the to them, of Biberstein to later vaccinated challenging capacity protective been development infection By injecting have which Deaths the after demonstrated was a most organism. against this and immunity "passive (1958) protected antisera, rabbit this of be to proved infection, of Smith passively has mucin haemolytica P. with study 48 hours septicaemia. be mice gastric for model within could with of with possessed antigens, and heterolo- and protection major role conferred, involved. in 37 a later However, 1969), in which similar a larger number original conclusions. antigens was two of animals, the antigenic there factors may exist somatic antigens typing, which P. haenzolytica days Three gations but anomalies were degree this pneumonia model be will that and immunity. of of mice with (Rushton, developed of shared laboratory infection been was capsular simple (IN) In suggested was specificity inoculation a non-fatal using by the found. strains the these capsular protection It than with some of the challenging other recently IN after of factors. intranasal has anaesthesia, of and determine. upon importance demonstrable for A model :doubt and Allison, undertaken The highest no demonstrable were cast immunising the when Biberstein experiments confirmed, experiments, shown (Knight, report 106'6-107.6 cfu, developed. Investi- described in later 1978). under this thesis. The and well pathology pneumonia of and sheep characterised. the sheep an effective to this vaccine bacteriology of associated However, organism against are at septicaemia P. haemolytica immune mechanisms with the both present pasteurellosis are of ill-defined, has yet and to be 38 The produced. into investigation models mice, of the of in to work described some aspects infection an attempt immune be response P. with to P. this haemolytica elucidate to of in the this problem, in is thesis both important an using sheep features haemolytica. 4 and GENERAL MATERIALS AND Bacteriological Strains All of strains A8, pneumonia. Strains of types in lambs, septicaemia of These were strains aliquots of aliquot was broth 370C. The (Oxoid* CM55, purity, and further broth strain Dr. H. Williams *Oxoid Ltd, T10 CM67) culture was streaked 15% citrated and from were type of in mastitis or in used as 0.5 blood to inocula of of Escherichia E. Smith. Basingstoke, coli, 50 ml agar check for It coli B188, was of Hampshire, was kindly bovine origin, England. ml an overnight sheep blood) A12 a ewe. into incubated onto ovine were A7, sheep inoculated and were cultures. Strain The of A6, When required, -70°C. storage colonies A5, strain of study freeze-dried, Oxoia* single this cases the and (NB - with A2, a case at from Al, T4 and either culture in from T3, from stored removed broth nutrient isolated isolated originally was at been used types of had All A9 and cases Strains origin. ovine tics haemolytica P. of 39 techniques haemol P. of METHODS provided by 40 9-, 078: K80: serotype (Smith enterotoxin in stored Viable Misra dilutions were duplicate 20 pl a. suspension in a colorimeter of P. and The standard were 370C (OD) the be the method of 10-fold L37) onto spotted were read whereby the estimated by UPS and blood sheep incubation after inoculated into for 18 hours, washed to cover were the also performed of (Figs. numbers 1-4). 400 on of of absorbance Each 300 viable These ml the NB and in 0.15 gave M NaCl, optical colorimeter. Viable measured. preparations organisms graphs strain of twice nm were these organisms measuring made which range at of number Photometer). was suspensions curves plotted devised dilutions of were counts was (Oxoid water these of (Vitatron at OD of coli Appropriate peptone counts could haemolytica densities in by out (1938). samples was in a range produce °C. 37 A method incubated E. not haemolytica. P. carried Irwin Viable at overnight were made plates. agar did bacteria of and but 1976). to manner counts invasive was Huggins, and a similar Enumeration Miles, and allowed. and. against inocula OD Figure 1 Standard A6, Figure 2 A7 and AB against Standard Al, curves of A2 curves and A5 of against P. types haemolytica OD at 400nm types P. haemolytica OD at 400 nm Co 0) ö Co ö a rö (0 8 cj 0 0 0 00 co öý 0 (n; o OI gold soli,cloweay "d ;os; unoo eIgeI rn ö d d ý c5 z a1 0 N co co (n; o 01 gol) eo 4AIoweay 'd 1o s; unoo ejgs! /1 Figure 3 Standard T3, Figure 4 T4 and Standard A9, curves All T10 curves and A12 of P. against of P. against haemolytica OD at 400nm types haemolytica OD at types 400 nm 0) ö 0 I- 0 M H CD ö 10 1 d ö M O ö 1111111 11 IIIII 11 1 IIIIi II1 1111111 1 I\\ CO (njo 01 eon) BoilAlowesy "d 10 sjunoo elqv , 0) ö Co ö NQ IDý ö co ÖU N Ö Ö (nia 01 gol) 13011, Klowee4 A io siunoo eI4gIA 41. to be stration; bacteria]. adjusted viable cultures to a determined counts were standardised prior density however in to obtained this manner. for adminiall 42 Laboratory techniques animal Mice Unless the of. were otherwise stated, mice used C57 black strain, bred at Mice Institute. old at the in housed (Laboratory for Oxoid of start Animal Ltd. ), sexes, They Diets, and were fed Herbert c. given experiments the Moredun and were Ten experimentation. cage. each both of were in water 4-6 mice Modified weeks were 41B Diet (Bawdley)Ltd., Styles lib. ad Inoculations 1. Subcutaneous into SC inoculations Mice anaesthesia. the flank, using injection per the flank, right a 25 gauge mouse was and Intraperitoneal Mice were needle. injected were injected were 2. 26 gauge mice needle. second IP Injection without was the under the required, the performed on the loose skin of When more than first given on using a was one left. anaesthesia, into without the abdomen, slightly 43 to left the 3. in (Fluothane, Mice were and droplets mice level of from removed rapidly, recovered of culture placed from induced usually Park, beneath wool insensitive recumbent, was Alderley cotton When the inhaled mice Ltd., ICI saturated jar. umbilicus. Intranasal Narcosis from the of narcosis a false was evenly the bell were on the anaesthesia jar, (0.05 external within floor correct, breathing saline inhaled Cheshire), and or halothane with held pares. a few a bell mice and ml), in were shallowly. upright which were The minutes. 44 Vaccines The this of various were work from prepared vaccines SSE or either in used much HKO of P. haemolytica. of strains haemolytica P. experimental Antigens 1. Preparation The provided by required strain into inoculated 37°C volumes this The 4°C. culture by The layers. cells broth was were were procedure kindly the of from removed for at purity Two pre-warmed inoculated each with agitation performed at solution removed and 12000 in resuspended and from storage, incubated and checked centrifugation salicylate This were 2 were An aliquot stage. incubated counts 1 and was culture this SSE Gilmour. nutrient broth Viable M sodium 3 hours. at and sedimented were 1.0 P. haemolytica nutrient 6 hours. at of identity of Chapters J. 50 ml L. haemolytica N. overnight. serotype of Dr. in used extracts P. of 300 agitated the and 1.5 with 15 ml 37°C at 1 for bacteria the g for 20 minutes sterile ml at cells. 370 C for the-outer 45 bacterial The at 40 minutes clarified by 30 minutes extract saline buffer (0.02 USA) stored until The of in pellet overnight. at and extract vaccines phosphateM sodium ultrafiltration Lexington, Corp., freeze-dried then and was deposit often present removed by the concentrate. the filtrate from in in 300 the scraping and and the cells 3 hours. The to concentrated A gelatinous surface with M saline M sodium 370C for at concentrated ultrafiltration 0.15 ml 5. centrifugation. morning, ultrafiltration. on The by following as before, approximately gently The 4 and Chapters sedimented resuspended 20 ml by in resuspended 40C. clarified was and was extraction used and shaken solution salicylate (Amicon salicylate grown, the sedimented were 0.03 by 20 ml, The against concentrated sodium were organisms stored g for SW24 rotor). 48 hours membrane was supernate 40000 at L, centrifugation required. slightly Thereafter The phosphate, approximately method -modified 4 C. for 40C, at X100A to 0 Model M sodium by removed centrifugation dialysed then a Diaflo Mass., at (Spinco pH 7.6) chloride; The 0 4C at was through were g for 28000 further cells the of a glass rod, SSE, were and each filter and was to added an aliquot dialysed of for 46 48 hours 4°C at dilute against then dialysed 72 hours. The dry weight determined, and. the extract and / matter ml, Preparations distilled against buffer phosphate-saline of each was using the dialysed were stored in (DW) for water preparation was adjusted to 1.5 filtrate as diluent. small aliquots at mg dry until -20°C required. Preparation 2. 1 volumes Two 1.5 as same manner organisms were in '1 r'iýy heat-killed of P. of in employed volume 7.2 pH and incubated tes. The preparation of haemolytica haemolytica the by sedimented P. were preparation 60°C was the The resuspended phosphate-buffered at SSE. of centrifugation, in grown (PBS) saline in a water bath for then checked for sterility 90 minuand freeze-dried. Preparation The antigenicity five adjuvants and Thompson, in either of has 1979). inc. 'mplete vaccines of been SSE of compared Sheep Freund's were P. haemolytica (Wells, Gilmour, inoculated adjuvant type (IFA), with A6 Burrells antigen complete 4 in 47a Freund's (CFA), adjuvant Mycobacterium of extract hydroxide gel adjuvant, hydroxide gel was vaccine induced against P. did than were not used in at the cation the aluminium at Alhydrogel each antigen Alhydrogel antigen (Table reasons, by prepared hydroxide, by antibody IHA an this most test local addition, vaccine the of adsorption followed antigens and of the onto HKO (10 gel vaccines the of by emulsifi- a titration was concentrations with 1). (A"ihydrogel, to according dilution, The Alhydrogel adsorbed determination When antigen optimal were adjuvant Alhydrogel, hydroxide aluminium mg/ml) after Denmark) recommendations. rer's of with hydroxide dilution optimal For mg/ml) Copenhagen, Superfos, inoculation of an aluminium onto of these of measured In last-named The titres aluminium oil. mineral (1.5 A6 as the which IFA. highe were Adsorption SSE into vaccines. For onto in other site study an aluminium a vaccine type this in or haemolytica of water-soluble tuberculosis, incorporated severe. antigen containing significantly any reactions IFA is of the mixed out a standard was manufacu- with flocculation carried the occurs. using a range amount diluted of 10-fold N H"ý" In to 00 H in LA 0 LA Un '-+ Un LA H LA d' " N d' O00 0 r-I of OD LA OO0 to OO0 In " OO0 t[1 in N LA IV! 000 lfl In r-4 " in MM ul LA MN ."a OO0 ". O00 " d' N LA d' LA "-I " H O" ßf1 "" OO N U) U) LA O U) M a oý H U) OO0 L en OOO U) N ý--I """ O00 rg a 8 wH H zwH H '- 0 4 47b DW for in this purpose After tubes. rapid temperature for in which series mixing, the taken tubes Alhydrogel Emulsification at in tube occurred, having for Wasserman left were first The as in antigen flocculation a marked of to minutes. was concentration added 20-30 supernate, clear and the that of adsorbed of antigen room the with a optimal antigen (Plate 1). antigen with mineral adsorbed onto Alhydro- oil The the gel at at room volume required This a fine A Arlacel that and the not a pipette emulsion 4°C during (Sigma was vaccine until Ltd., in emulsion If the desired storage, and it was ensure adequate a syringe volume USA) St. a tissue homogeniser. was the Louis, a water-in-oil vaccine drop type. necessary fitted of sterile 10% containing Company, On occasion, required. and equal emulsion, water. of an left prepared, to from Jersey, Chemical an oil-in-water was into New resultant onto forced. was emulsified was 30 minutes needle gauge (Esso F Bayol for temperature flocculation. with concentration optimal USA). The To check emulsion, was dropped remained discrete, Vaccines were the to emulsion from the kept at separated re-homogenise the 48 preparation. Bovine In bovine optimal With Arlacel (BA)(Bovine albumin Ltd., Aihydrogel an equal A. not by was made This Laboratories did which vaccine antigen. it experiments, several placebo vaccine albumin volume concentration, of a solution adsorbing (Fraction England) sterile onto and Bayol use a haemolytica P. any contain albumin Slough, to was necessary V) 0.1% of Miles Alhydrogel, emulsifying F containing at this 10% the 49 Techniques by employed transformation Lymphocyte collaborators to responses haemolytica p.. antigens Lymphocytes blood Uppsala, Chemicals, Fine 1.075) at (HBSS) Solution 1x resuspension 10 5x Twenty to SSE was added for of and wells 20 p1 of lymphocyte Plates suspension All cells/well). were and sterile incubated was cultures at Balanced heparin of ml in medium P, 370 C in with plasma. set each up a moist well in type Al microtitre to 200 to medium haemolytica added cultures. were 1640 RPMI flat-bottomed sterile added before 20% autologous of were Salt supplemented a dilution of which interface, Scotland) control non-stimulated / cells 2-mercaptoethanol microlitres plates, 10 Paisley, (Gibco-Biocult, 5M 6 g for lymphocytes, Hank's - gravity 500 at The with 440 (specific fluid 5 iu/ml containing at (Pharmacia Ficoll/Triosil-plasma 3 times England) (Tridsil mixture unmixed of Liverpool, a Ficoll of temperature. washed and removed Norway) the at a volume Medical, volume the room a band Evans layering Sweden)/Triosil centrifuging and 30 minutes formed Oslo, & Co., Nyegaard BP, an equal onto by obtained (Heparin heparinised sheep were pl wells used of (ie. 2x triplicate. atmosphere for 105 5n in 5 days tion of the (specific Amersham, each box. a sealed 1 PCi cultures, activity 5 Ci/mole) England) in the At well. end glass-fibre automatic cell harvester in were and vials Lymphocyte counter. P. type haemolytica RPMI of the in an were Centre, was added to cells were period, with Abingdon, 1 ml termina- H,Jthymidine medium discs a semiEngland). fluid scintillation liquid automatic proliferative Al ? culture (Cryotech, in before (Radio-Chemical of immersed counted hours m3thyl filter onto discs of 20 pl adsorbed Dried Eighteen scintillation responses expressed as to stimulation (S. I. ) indices Counts S. I. -3H-thymidine containing due to incorporation minute into lymphocyte culture R. haemolytica antigen Counts per 3H-thymidine minute into per of _ due to incorporation lymphocyte unstimulated of cultures Histological Blocks dehydrated Sections eosin examination were in were (H. E. ). of from prepared mouse lungs formol-fixed alcohols and embedded prepared and stained in with tissues, paraffin wax. haematoxylin and 51 IHA Serum was in 1975), incubated added at 37 formalin), 0C in 3 times washed with to volumes added to each 2 hours, test sera U-bottomed ml) the well, then 5%. in at at of solution. in buffered microtitration was 40C overnight, morning. FXBißt left bovine and then plates USA). Virginia, at And (0.3% same made sensitised j 0.5% RSC were saline the RBC, Osterman, and bovine Alexandria, test W co were The of formalised Company, (0.025 Dietel The culture, Bovine concentration 1 hour. of saline Equal Biberstein 18 hours. 30 minutes. (Shirai, buffered in for for a final for Engineering (Cookes 0 56C resuspended and formalised sera antibody broth nutrient agitation at dilutions Doubling from 1% gluteraldehyde, were sheep (Shreeve, test IHA for tested were an prepared was heated fixed for 370C at culture sheep by in antibodies 1972). Antigen incubated from haemolytica Thompson, and haemolytica P. samples P. against for test room read RBC were temperature the following 52 Challenge SPF lambs of P13 virus with and P. haemolytica This duced in pneumonia been in reported Thompson Lambs after an Lambs inoculation each at P. haemolytica, abnormal P13 and IN and Smith, in maintained McVittie P13 with (1971). (Wells, virus 1976). Seven lambs (Gilmour, isolation Mellor and the virus, examined were the the awarded IT P. haemolytica were days exposed to Smitn Thompson, same to clinician group time each respiration To avoid was of lambs day each unaware prior following a score for lamb. Dullness, were 2 weeks for clinically P13 virus. with influence, given Gilmour, 1975). Angus, to and Mackay, with of aerosol and delivered Rushton infection (Sharp, has and 1978). Hart, Burrells, lambs of elsewhere in- consistently proportion detail inoculated were Sharp, a high were by described infection challenge Rushton, and SPF lambs as of method the assigned subjective any of to treatments the and . challenge, the degree of aerosol of illness (> pyrexia one after point was 40.60C) each, and and 53 death four signs of Lambs points. Pathology Lambs days killed 7-10 were submitted dorsal the lung as a percentage Sharp, Rushton, scorc- :: crc At the of between differences the of Portions sectioned samples of for of lung the groups test Mann-Whitney added were (Snedecor lung were. histopathological tissue basis from 1978). of area daily Lesion >25% by means Cochran, in = 20. scores formol-saline and and of lesion the 1967). examination. lesions thus: affected, (clinical determined fixed expressed Statistical scores and on (Wells, clinical together. and recorded and area = 10, the total were surface 5,11-25% experiment were animal each the on were Lesions lung Thompson, were haemolytica a planimeter, lung and < 10% the showed animals P. the of with total Gilmour no scores) measured allocated ill examination. aspects the when surviving with post-mortem of = 0, all challenge ventral lesions for after diagrams, on killed were Also, for and end or necropsy. at examined died, they Bacteriology and which if disease. respiratory severe killed were "normal" and Further lung - 54 tissue macerated were (Colworth) by the the and Quantitative in sera technique (Voller of Engvall p. haemolytica pH 9.6, counted (1938). P. hae molytica to immunosorbent 1975) SSE Plates 0.05% between M129A, these in PBS/Tween added in 300 and µl 20 to testing containing washed and 0.02% to at the the room wells of buffer, polystyrene Laboratories wells Reference technique dilution of held 3 times at out test dilutions sodium azide of temperature 4°C excess sera 1/1000 with PBS with shaking and Ltd., filled were plates (PBS/Tween), wash. volumes incubated wells Dynatech and modifi- ELISA M carbonate Individual Tween prior 0.05 the then the A 1/100 sensitise were each pretreated in a microplate of (1972). solution, antigen of basically was (Burrells, test serological. Sussex). containing plates, this (type plates overnight. not haemolvtica antibody of Al to used Billinghurst, fluid Irwin a micro-enzyme-linked Perlmann type was pl and Bidwell, and and microtitre 300 Misra by 1979) Dawson, and cation P. a homogeniser (ELISA) assay Wells viable of in water of estimation sheep The peptone number Miles, of method with were of were antigen-coated for 2 hours. 55 Serum dilutions were then washed 3 times with plates in 1/1000 PBS/Tween 3 changes with again (p-nitrophenyl Dorset) a concentration (B. D. H. 1*I 9.8, (300 the wells the reaction 0.5 length of 400 of fitted on a chart substrate Company, in Poole, 10% diethanolabuffer, mM magnesium chloride was After by 1 hour the an X-Y included the serum reference processed to a at addition of to added temperature, room 50 pl as of allow in digital 3.0 M the end a computer for drift of test, each the were of reference each whose Photometer) which by on a punch-tape standard of a wave- Results co-ordinates form of at UPS cuvette. the peaks, was. read (Vitatron flow-through beginning at samples colorimeter Five plotter. at individual into translated values in nm a micro with recorded was washed Dorset) stopped Absorbance of and Poole, µl/well). was 3 hours hydroxide. sodium were Ltd., for Enzyme Chemical of diluted IgC emptied 1 mg/ml of Chemicals containing were Sigma pl incubation PBS/Tween. of phosphate, at mine wells the wells, 300 and After the the anti-sheep added. temperature, room at PBS/Tween, phosphatase-conjugated alkaline from aspirated run. programme baseline were sera and means a further The data tape adjusted during the reading of 56 the measured samples, reference relation sera. to The a calibration by in variations results curve were the finally provided results of expressed by the the in standards. 1. - Chapter infection Experimental P. of mice with 57 haemolytica Introduction The The of this of screening of which involved factors the (see a method P. haemolytica, with and in described work development of discussed been have in potential of to the field of the organisms establishing disease of within of mice General chapter was be immunity aimed P. in have haemolytica a model features with host, resulting are described mice investigation Ideally, pasteurellosis, the the at for used to should haemol- P. with infection challenge could and Introduction). vaccines. infection haemolytica P. with infection experimental evaluating ytica associated problems similar multiplication in a true infection. Two models In the of the first, second Smith is of infection P. haemolytica was. given a modification of the IP in this chapter. intranasally, challenge and method (1958). 4 58 Materials Strain P. haemolytica strain of of The is ments Materials General Intranasal 0.05 prP ml of ration washed in resuspended bacteria of Enumeration and Methods. of PBS for described strain of in were PBS by give an 89 10 -10 for P. detailed are mice Each received mouse IN. intranasa. in PBS to viable experi- Control mice IN, cultures twice inoculum Al suspension inocula between the comprised inoculation sterile broth Nutrient 5 hours, IN P. haemolytica 0.05 received and Materials of ml type these procedure challenge General in used Methods. and Anaesthesia in haemolytica P. P. haemolytica the Methods and ]. challenge incubated at centrifugation estimated cfu/ml. 37°C and concentration This for of suspension mice. haemolytica in lungs of infected mice At selected time intervals after challenge, infected 59 and control lungs their 9 ml Stomacher, dilutions counts performed lung in ogical for was Laboratory, Seward were by removed from enumeration the of bacteria. (Colworth water, Miles, a small diaphragmatic lobe the and Misra 4, all with Appropriate peptone of and maceration a homogeniser 1,. 3 and before examination, in method Experiments to London). made the dislocation cervical prior in water peptone 10-fold (1938). aseptically removed sterile by killed were mice remainder viable and sample Irwin of for histol- was macerated 60 1- Intranasal varying numbers Experiment Design A series 7 groups and are maximal the basis of mice group of score lungs of the this 1+, lesions in were Swiss mice white this according and Each to lung the no and was extent the third samples from lungs On Materials General attempt group the on removed the which 1978). killed (see experiment, assessed, challenged mice in were examination the shows (Rushton, were were mice, group. lesions mice individually. , -) each challenge these PBS was made, 1.1 Table by that after of in dilutions. evidence, lungs Methods). 2+, with described as 6 C57 black develop histological for taken (3+, 3 days The day. shown prepared of received haemolytica P. with and these been has It mice haemolytica dilutions consisting each organisms of number P. was 10-fold of IN with infected IN inoculum challenge above. of of experiment of The infection each was made given and to a score severity present. 4 1.1 Table Degree No Group No of type P. haemolytica Al given 7.3 1 10 2 106.3 10 4 104.3 3.3 5 10 6 102.3 7 101.3 in lesions 3 days after 2+ 2+ 5.3 3 IN of of 1-+; 1+ severity the lungs challenge 61 Results The of 3 days mice 1.2 Plate degree been of the air In contrast, is shown pneumonic, bronchiolar the higher from shows alveolar septa teristic of severe dation, Plates 1.6 of the - 1.8, in in seen exudate hyperpl&stic illustrated. is by of most which epithelium, swollen. lung which scored area of 3+, lung the is hyperplasia of Some sloughing. - illustrated 1.8. at of 1.5, swollen cells, charac- pneumonia. More with 'and pneumonia consolidation, 1.4 Plate illustrates macrophages and that are mononuclear extreme seen a septa 1.4 Plate of partially are and be lung and focal 1+ severity with an features The are pneumonia and 1.2, Plate 1+ scored can and Plates infiltrated lungs large 1.1. (1978). the in group size pneumonia in magnification detail it reduced with this of to consolidation epithelium features of-the . A focal with compared affected 1.3. Plate which each Table Rushton interalveolar a severely in by 1.1) are the that and collapsed, with spaces in shown a mouse (Plate mouse alveolar of When pneumonia. sham-inoculated is described lung the shows in observed infection after have lesions The lesions the of severity focal consoli- neutrophils. are shown in bronchioles a necrotic small artery " Plate 1.1 Lung IN. from C57 black Note expanded saline given mouse alveoli with thin H. E. X76 septa. Plate 1.2 Lung from C57 black P. haemolytica The swelling Al spaces due partly lung type alveolar to of but interalveolar degree in reduced are 1.3 Lung The focal showing main 3+ changes also 11his septa. severity- of degree are consolidation, bronchiolar sloughing epithelium size. to X76 H. E. Plate 1)" (Experiment IN collapse, 1+ rates given mouse ý3 cfu lÖ severity. of pneumonia and with Of hyperplasia with focal (arrows). H. E. X76 S J Plate 1.4 Higher magnification in strated of 1.2, Plate interalveolar showing cells, cells, are type possible also by. Some hypertrophied cells. lining swollen infiltrated septa mononuclear illu- section II alveolar (arrows) present H. E. Plate 1.5 Pneumonia associated P. haemolytica ment 3). with an with type There exudate is of Al 106.8 given focal X875 cfu (Experi- IN consolidation, macrophages and neutro- phils H. E. X875 Plate 1.6 Severe consolidation the has of in lining a hyperplastic process cellular bronchiole A respiratory 'exudate. (left) with sloughing H. E. Plate 1.7 Bronchiole hyperplastic with X87'5 epithelium X875' H. E. Plate 1.8 Necrosis of of consolidation. lymphoid cuff the artery :. uclear cells of small artery A narrow has is formed, infiltrated an area within perivascular and the by H. E. wall mono- X875 62 Tt the is evident lesions infected Al fewer of did not No bacteria 3 days they Experiment been were 104.3 than 2 was the severity in the of organisms. P. cfu lungs of of mice haemolytica lesions. produce cleared that numbers in observed This infection. after had largest the with 1.1 Table was greatest observed Inoculation type from from designed suggested the to the lung investigate by lungs that this this of Mice perhaps time, and possibility. 61 2- Experiment Viable counts lungs It from to lied important was cleared after discover to infection whether bacteria of challenge, infection. a true which of two the were if This these in haemolytica P. after mice know to lung the establish designed of of they multip- experiment was alternatives occurred. Design of experiment Fifty mice F. haemolytica of 10 mice In ted were the second 107 with of groups 48 hours on were the killed and the this the time 72 hours of after 50 mice Al challenge and and Viable mice. were killed infecin 4 hours, counts 4 hours, Viable individual of type later. and challenge experiment, of Groups challenge. lungs haemolytica cfu previously. time 16 hours on of P. cfu the at 107 IN with described as performed 10 at and Al, part inoculated each type 12 hours 8 hours, counts were 24 hours, were performed lungs. Viable and standard counts error were expressed (SE) calculated as for log10, and the each group. mean 64 Results The It Al type the viable transported are within bacteria shown in the are Fig. lungs present 1.1). situ the 3 days in the and 48 hours from to the lung, Lesions are infection, lungs that impossible within lung. after but free is killed are from it 1.1. haemolytica substantially data Fig. mice, in killed are bacteria the infected of this P. of or lungs the From whether present (not removed haemolytica. they lungs the are challenge tell if in in graphically no multiplication occurs organisms after P. that appears depicted are results at yet this time or no Figure 1.1 Viable counts in lungs, the of of P. haemolytica mice after IN type infection Al U 0 E 7 C) co 6 L 0 5 4 0 4c3 0 U2 1 co > 01L1VLVL Hours IF after infection '{ a 65 Ex eriment 3- sodium Comparison Experiment anaesthetic 3 was by given inhalation infection discover to performed thiopentone and intranasal for anaesthetics as ha lo thane of if the for was. responsible the lesions. Mice it is after were the of ca type groups of 9 mice lesions seen and may be partly in lungs the this or possibility, with either halothane, IP, before IN or infection with Al. experiment mice 9 mice in infected described anaesthetic To eliminate given sodium Twenty-eight and (Intraval were two 5 mice of groups Group anaesthetised IN with 106.8 P. The by IP injection sodium: cfu of May and each with haemolytica 9 mice to allocated randomly 1 were previously. anaesthetised sodium for anaesthetised haemolyti. Desicn and this infection. thiopentone The that responsible 3 days p., exposed conceivable wholly mice are inhalation, by halothane to in 0.2 Baker Group ml two (Table 1.2). halothane, type Al as 2 were of 10% thiopentone Ltd., Dagenham, N p tn 0 0 0 r- 1 0 0 H N M M to to N "9 0 0 H H 0 0 0 N r-1 M O 0 O U (n rZ W I~ O "r4 to 0) r-+ 1 r-I . -1 (0 r,U S-+ 43 4-1 4-1 O (U U) "r4 1-I +1 + (U 'ZS M %U "rl M N N 0 0 U to U (fl U .ri N d1 0 N ".-1 d) 0) 0 4-1 N 43 Co a) r4 c) r" 4 41 ä ä0 0 0 01 -r1 ., rcj U) Z: r--1 A A o -r1 0 rd r-i ß u0 (Ti 4i , H Ch rn In In ä Z N ° " U) Hc) "3 0 2 u u "r1 3 -,-1 e -s- aý .-4 4(0 ;I 40 (1) . --1 N M d' Z 0 ic 66 Essex), infected and Al. type consisted thiopentone and inoculation to 5 mice, of halothane with prior days Three this 1+, The with 0.05 were killed, ml comparisons mice group were to 3+ were 2 points, awarded + were given to calculated, the the represent scoring was assessed scoring 2+ were be made between to mice Mice and each histological a score scoring 1 point, for total for individual awarded those mice lungs lesions. of 3 points, scoring 0.5. and severity all their from experiment, awarded those from Lungs and extent infection, after removed samples examination. in each haemolytica P. cfu PBS. sterile and 4 anaesthetised respectively, sodium 10 with 3 and Groups were which IN 6.8 different enable groups. Results The The than mouse), both Lesions negative usual and Group in Experiment inoculum challenge lower of results (10 in griups (ý), and cfu as Group experiment 1.2. Table was P. haemolytica a result 2 showed 1 and the this in shown slightly 6.8 probably 1 and 3 are 2 varied spread was this, of no type three lesionsain from similar Al in their the in mice lungs. (3+) severe very per two to groups. 67 Control It can is thane given mice be since challenge, anaesthetised similar Group not 3, mice with lesions to which were infected, from concluded responsible not PBS sterile the the thiopentone mice in no lesions. experiment that lesions same Group lesions. 1, 3 days (Group and with the halo- after but challenge, sodium anaesthetised displayed no showed this for given IN 2), mice halothane, had in but 68 4- Experiment Comparison of heat-killed bacteria No detectable when lesions lung the of These anaesthesia. suggestion presence lung, large of and present of the Experiment not a direct led the bacterial result to the the of result in may produce to test lung 3 material, designed and mouse and may be 4 was in observations P. haemolytica Experiment results. two amounts dead that are were lesions the that haemolytica present, live with P. lesions the that demonstrated are infection IN the similar hypo- this thesis. n of Desi experiment An inoculum as at 600C for of both live killed and to heat-inactivated were the the prepared (10-1 determine check was 10-2) and made, and number sterility of of the heat- preparation. Forty-two of inocula to performed and was Ai Two dilutions heat-killed present, type One half 30 minutes. counts organisms haemolytica previously. described viable P. of 6 mice each. mice were Mice randomly were allocated anaesthetised to with 7 groups halothane, 6 inoculated and P. IN with haemolytica The seventh The treatments at live either 10-1 undiluted, group inoculated was to given each heat-killed or 10 or with group 2 dilutions. PBS. sterile in detailed are 1.3. Table Three days after samples of lung examined awarded as in the challenge, mice histologically. Experiment killed were Scores and were 3. Results The are inoculum live p. results that was type haemolytica in shown to given Al/mouse), severity observed in group. Mice in 3 were five of bacteria the six groups showed either live control showed Group no no (Group in the of were mice positive were given varying in this in these two inocula, The results. sterile of in present Smaller few cfu numbers also lesions. lung. mice equivalent remaining -which of six 6 (10'2 6 Group of the largest The lesions given produced 7), mice lesions convincing dead, lesions five The mice. or mice and 1.3. and were heat-killed Table PBS, 'n ýý 0UN U) r' L4 N90 Ln 0e Ln r-I O. ý-+ NO0O to OOM0 ANIM r-i to 93 O "r"I W u >"++1 4) U) N r"1 4-t fCS 4-1 to O oO00 O ýn !6 '-+ NH rl 41 M ri 4i I-ý N fn N + N: ri N0000HN0 N N M r N il Q z3 ý 't3 ý r-i r-H r-H r-I r-I . r-I d N ;3 r1 Q rtf u "r >1 Ho Q H 0 *H N N 4J w CJ "'i 0 U) 0 U) "ri "ri 4J aý U) :1 U) ni N N (V (V N N d' LO ýO 4 in 0 O "I w 0 (V O O 0 0 0 r4 r1 4J tn 0) 0 bl r3 u b U rl u 0 +J z w 0 ° z 01 0 C9 4) r-I .Nm d' LO 0N z 70 It 3 days but are material or probably in the with heat-killed the due merely to lung. that experiment in lungs the inoculum an P. not are this produced challenge lesions these be can live from concluded IN after either that be lesions similar of can haemolytica result the of presence of mice 10 of type ongoing of 6'2 cfu Al, and infection, bacterial 71 5- Experiment Intraperitoneal inocula of preparation challenge for IP determination Design of by volume 10 hours were pended 5 1-10 at 1000 g for 20 minutes washed twice in from saline were 7.2 to 4 parts inoculated no was IP deaths further calculated (ICN gastric recorded deaths by of 48 hours occurred the and method dilutions and a viable and dilutions of Groups later. after of Reed 1 part 5 mice of and the Generally, time. and hog Life dilution each this of Inc., a ratio The resus- a 5% suspension at ml 40C. 10-fold of mucin. 0.5 with M saline, broth obtained at Pharmaceuticals NY), The bacteria the suspension into Plainview, bacteria number and a fresh Al. suspension, undiluted incorporated Group, of this The pH 0.15 A series saline. Sciences were type and mucin, gastric mice from 37°C, performed. count inoculated at 10 ml made in was of LD50 haemolytica P. of centrifugation in of NB was of culture for bacteria 10 ml agar incubated challenge experiment A 300 blood procedure; The Muench LD50 (1938). was 72 Results was In the than The LD50 105.3 cfu subsequent inoculum the LD50' of this when given experiments was strain always of IP in with greater P. haemolytica gastric this by type to mucin method at least of 10 Al mice. challenge, 0'5 cfu 73 6- Experiment Growth of and spleens livers This organisms experiment was multiplied within a true infection. Design of as of in the described and by P, IP gastric mucin. 8 hours after challenge dislocation, cervical viable were in if the mouse the mice established and with 10-fold 107.1 with At peptone dilutions The counting. calculated and The counts Al was in their organs in counts expressed as of were and were of haemolytica 5 mice livers peptone mean P. intervals of water prepared A group cfu time groups These 9 ml type experiment. and aseptically. macerated serial the Al determine to haemolytica in removed normal designed preceding inoculated was Al type of type experiment An inoculum 20 mice P. haemolytica 1,2,3 killed spleens indivually a homogeniser water ± plated SE for log10 each and for out group cfu. Results livers viable and spleens of of mice P. haemolytica are shown type in Fig. Al 1.2. in the Each Figure 1.2 Viable counts of in livers and the owing IP infection type P. haemolytica spleens of mice foll- Al 8 U O E O Co O Cl, .r C 0 U " " G) "--" Liver ....... Spleen co i 2345678 Hours after infection 74 point plots spleens, livers after with "true" the the mean similar infection organisms are spleens infection, SE of log10 at expressed and ± the growth was within organisms in taking the counts Counts cfu. given rates the host. in Appendix multiplied each. place, This with for 5 livers from 1. in or individual From both showed multiplication 2 hours organs, that of 75 7- Experiment to prior This model Design of mice each. in Those inoculated onto and Group Alhydrogel routes type in 1 were inoculated be used vaccination to with an the ascertain mineral ml of 5 were and this vaccine in in emulsified in after vaccination, gastric General mucin were controls. ml of Alhydrogel in Group IP. ml of of 4 were Mice in BA adsorbed mineral Details given 0.1 mice 0.1 given of unvaccinated onto oil, 5 groups to SC with SSE adsorbed respectively. weeks Al Group Al and are preparations tica the vaccination. Mice 0.1 with Three Al if could and allocated in 3 and IP by randomly type emulsified Group haemolytica were 2 were Group haemolytica and type experiment Fifty 10 mice SC routes and determine vaccine, for route suitable most to afforded haemolytica P IP haemolytica P. P. with by mice designed protection experimental of with was infection demonstrate to P. challenge experiment Ii` of Vaccination by oil, these Materials inocula prepared and of the Sc vaccine Methods. P. haemoly- as described 76 for IP previous were group 10 5 with IP challenged 7'6 Five challenges. The cfu. with 108 number of the of 6 cfu in mice the and deaths each remaining recorded was later. 48 hours Results 48 hours Deaths 10 control All P. vaccine 107.6 cfu, but 108.6 cfu died. number challenge In contrast, and haemolytica mice survived challenged IP complete were the lower with inoculation protection lo6 the five in IP) 4 (vaccinated both IP and with four 3 challenge of the with higher the given (vaccinated with with Group those all survived challenge in survived Group challenge challenged mice 1.4. Table SC with from mice in shown vaccinated but organisms, 5 Group five BA vaccine) mice are protected the of vaccine in Mice Two of of died. P. four SC with (vaccinated challenge died. mice haemolytica smaller after with challenge doses, BA vaccine) all the five mice 8' cfu, of from survived. the P. haemolytica both challenge vaccine doses, afforded whereas only 1.4 Table The 'of effect vaccination challenge with by P. adminisVaccine No 1 * tration P. 3 BA* 4 P.. 5 BA Both haemolytica* haemolytica antigens emulsified adsorbed in mineral of type of hours after groups deaths 48 challenge of cfu "108'6 IP Al Number in upon five mice 107.6 cfu - 5 5 Sc 4 0 Sc 5 3 IP 0 0 IP 1 0 - 2 SC routes and haemolytica Route Group IP onto oil. aluminium hydroxide and 77 one mouse However, survived. IP was at the have with the to reasonable IP to shown vaccination with SC with vaccinated conclude administration least in part, peritoneum. vaccination subsequent was the a protective haemolytica that to For this in the the some vaccine the of effect haemolytica P. due experiments. P. adminstration of chosen the BA vaccine demonstrated protection haemolytica preference in reaction the SC route to is, vaccine non-specific reason, is It vaccine. P. to comparable the IP of route in IF 78 8- Experiment The type P. haemolytica in organisms effect Al type IP 10 with Five mucin. time the with growth of with challenge later 3 weeks Al P. cfu challenge, removed these challen- were killed thereafter. counts viable and ml gastric 6 hours and aseptically, in Al mice 2,4, and were type 5 control 0.1 later, weeks controls haemolytica and SC with Three 25 uninoculated 6.6 were inoculated each vaccine. vaccinated of livers Their were Al of a group and ged at mice haemolytica mice IP mice experiment of Twenty-five p. the upon following type of vaccination vaccine liver the P. haemolytica Design of -determined. Results Viable of and vaccinated Each given are as log10 in Appendix in exponentially there in the was control the represents point expressed P. of counts a reduction livers of mice mean 1 1. Viable livers of in vaccinated the type are shown SE of from Counts cfu. the haemolytica of Five livers the 1.3. Fig. from individual untreated mice. in counts counts number in Al 5 mice, livers increased mice, viable control whereas bacteria mice Figure 1.3 Viable counts of P. in livers of control mice the following IP haemolytica infection and type vaccinated Al Co U Ö E cd Control 0 "" "" N C ö5 U a) 3 > 4 i i "''''""""f 12345678 Hours after infection Vaccinate 80 which . were not sacrificed contrast, 5 infected time, P. livers. and haemolytica all vaccinates could died 19 hours. with appeared not be healthy recovered In at from that their 81 Discussion The development the was challenge in in which the in IN of lung, the seemed the primarily during result of was no net multiplication of by n8 hours a.ftcr P. haemolytica in which (1978) P. recovered some mice of by haemolytica from individual evident from not and not within multiplies faster any lung, this rate, or removed pools and the that of lungs in this it. is Lb e lung, from viable 5-10 mice, It experiment. haemolytica whether removed from former from with study, are it cleared may be killed The there numbers P. bacteria Bacteria from varying In as probably ruzhton challenge results lung in by caused infection. after method. whether multiplication. or 3 days involved However, P. haemolytica mice, the study promising. not so were bacteria. the of these were challenge, upon performed were counts is cleared lungs the effect organ these and the was the infection anaesthetic an chapter for suitable the used this preparations. pasteurellosis, pneumonic in P. haemolytica of be would model developed Lesions a method vaccine first, At described work of mice experimental of the of purpose within possibility a at without the is 82 the more likely. were mice when Green Staphylococcus aureus of disappearance of their by 80-90% of in only that mechanical in viability as rapid, removal the total it but clearing is declined these that probable small decline lung mouse methods by indicated rates The the rate declined a relatively in rate the with viability process. haemolytica P. of compared between comprised the and radioactivity disparity The of whereas radio-labelled mirabilis, bacterial removal, that showed of bacteria viable 4 hours, 14-20%. an aerosol Proteus or mechanical fraction to exposed (1964) Kass and of is not removal are similar. The the is fact mouse lung fact the The "the and cheaply P. haemolytica extensive mouse induced, in ch_nges to therefore infection (1978) besides provides have with of being lung and subsequent without the death". be important living not viable this quickly, an opportunity the more are claimed in multiply do not to pneumonia, not but lesions solely Rushton haemolytica. does disadvantage, bacteria the related that haemolytica a serious lesions. necessarily P. P. is that lung cause that for development Despite model easily studying of these to 83 advantages obvious of it lesions, to was therefore turned effect this In host IP lesions and for the study of study of route, were that the of which model haemolytica P. immunity. infection death Attention with mice developed was by not quantify the number the differences survival may be, the the data face of 100% of of, increased an for of even agent death, to greater has reveal by in of been enhanced present. if death the by or of host. the Obvioumice, vaccinated represents the a interpre- vaccinated mice, difficult. more problem the but of mice, nothing controls, protection, original of survival between a group is his organisms 50% survival example, time of kind 100% mortality demonstration convincing this In or inactivation of survival death of bacterial of machanism infecting 48 hours. recorded significant becomes within the within multiplies (1958) However tation haemolytica Smith did sly, P. model, causes and work, or the the bacteria, of by that experiments the to model the (1958). Smith in of the the that induction of rapidity and showed particularly haemolytica and study unsuitable infection, P. cost, considered this a toxic due was was in described low of This virulence some form of of the immuno- 84 Serial in both these enumeration of growth Such is which may be are studies Detailed of P. in ated CFA, in the lower livers than and in administered highly spleens prior considered of to be of to greater actively the Extensive with performed growth these value nature 1974). Collins, and against P. haemolytica of both parasites, incorpor- parenteral P. of vaccinated of data. microbial mice mouse SC challenge, view bacterial disease. vaccine, Hyperimmune In viable been absolutely controls. IP of (Woolcock and the the prevent revealed heat-killed multocida, protective. enumeration was P. with challenge have of protected in (Collins, mice of Two injections 1976). a number nature infection inultocida shift using have animals to estimate mortality/survival obtained species a similar of studies to some of to to sufficient curves response Salmonella including a significant immunised immune circumvents used complementary growth passively the criterion vaccines rate, mice bacteria of populations control The criticisms. the of and vaccinated efficacy and host. the of suppresion was multocida was 100-fold serum, shown to be experiments, in challenged than mortality/ mice 85 data survival An the of aim P. haemolytica by at in between differences at time of This after. screen each gation of far vaccine of LD50 P. haemolytica degrees stimulation following as the serum convincingly it for be would each there- for investiThe scale. would vaccine to required require animals. BA vaccines administered of protection (Experiment 7). of peritoneal macrophages can mineral only interval time one was group each on a large and introduction and be from mice successfor an advantage values of The specific fewer preparations numbers similar at increased sufficiently to mice preparation, vaccine of experiments, kill vaccine had livers the groups and that greater gave such challenge, meant determination two to in a viable mice control decreased subsequent sufficient considered the had the In demonstrated. of of potential infection, after those and development was screening livers mice vaccinated for hours the 10-fold, least study used By 5-6 preparations. fully present be could which model alone. of oil, a variety *into the of IP Nonoccur substances, peritoneum (Stuart, 86 Habeshaw Davidson, and took stimulation the reason, IP route demonstrated serum, lated IF, saline mucin. gastric doses in Protection or then P. effect haemolytica of study results are administration by agreement. of the only fewer of vaccine. either aluminium sheep Smith formol-saline sulphate, the was used also vaccine strain of isolated pneumonia, (1959A) a single flocculated vaccine strain of by given experiments living of haemolytica that the from mice inoculation Although and lower at deaths of with to oil. a case from IP P. potassium these inocu- controls. formol-saline in diluted haemolytica P, evident vaccination mineral (1959A) were protection superior septicaemia, was in IP and used lamb in than later Smith if with when that in effect was haemolytica the in that challenge haemolytica, bicarbonate present IV This was with emulsified a case to by preparations note buffer a formol-saline afforded SC vaccination prior P. avoided such for and was veronal-saline demonstrated with challenge sodium to that possible protective mice- (1959A) P. ' haemolytica by interesting P. of treated Smith these experiments these is vaccination or hours 5-6 challenge IP in a non-specific mouse occurred It of is It experiments. in place 1978). from in the these found gave that a R7 degree similar oil an was The challenge to the and of the in use field the used lambs in this which model obtained favour P. that and that suggests of P13 virus in P. with 1979; there may be the haernolytica an infection two infection can be comprimised, infection do to type (Gilmour, Chapter some Al protect haemolytica in the 2 of established, SPF Al thesis), this between This systems. has vaccine Martin, correlation mice those resemble type shown challenge also However, method infection Wells, in 1978) before haemolytica been its enhance be must sheep, P. haemolytica, Rushton, and this resemblance pasteurellosis host P. also challenge on by and in of established. The has study Thompson results lambs disease. against superimposed Sharp, in produced no to infection, be can inoculation. bears mucin Thompson P13 virus IV host pneumonic the for study pasteurellosis gastric of present it that a natural that haemolytica lesions of model by is the SC immunisation the pneumonic of Gilmour, case P. in of not disadvantage this with of the (Sharp, the used it when achieved unsuitaLle model is The virulence. has mice mouse necessitating sheep and disease field that disadvantages major that vaccine emulsion, IP to protection The IP. given was of in its and its 88 progress followed Further procedures. described in in later the face experiments chapters. of various using vaccination this model are Me.^hanisms immunity of 89 2 Chapter to haemolytica P. in sheep Introduction Assessment has previously IRA test been to P. (Cameron. lambs to evidence P. to titres of ability been been has antibodies suggest that capacity Al did not the of antibacterial combined and phagocytic in the these of of humoral is not lungs to passively ' P. The has the and resistance haemolytica these type parameters. immunity in protection clear. is normally of the Inhaled systems. regions the lambs of Al given with either activity immune upper sera of Correlation titres type challenge with the IHA of antibody indication disease. serum a lack serum communication). to role is of any prevent of vaccinated There measurement personal correlate Sterility impact of P. haemolytica against 1970). The criteria. from sera vaccines detection the haemoivtica P. lambs Consequently, in between protective for gives to vaccines serological used haemolytica (Gilmour, upon haemolytica P. of haemolytica Smit, against of based widely demonstrated individual efficacy and vaccinated mice the of of the maintained by mucociliary, bacteria respiratory which airways the 90 are (Kilburn, systems invading 1967; lung the by entry in inactivation in mice, and Green 1974; isms is known the Two experiments first was ytica by and absence experiment, serum prior experiments would in be immunity guinea are lung sheep of lambs to gained to with in the (Goldstein, Green Warshauer, in local defence during of lambs. in the In with haemolytica. the hope that which sheep. mechan- The chapter. in vitro, and pasteurellosis. this immunised P. 1973A Green phagocytosis mechanisms haemolytica extensively and in immunised performed P. lung passively challenge into sheep cells from were were the involved been and about into' wash serum Jakab described investigation an pigs Lippert events has Lippert 1964; in operate which lung the their after (Goldstein, sequer. ýe of Goldstein, Kass, and Little 1973B). and bacteria and minutes (AM) in rats 1970; Seamans, 1968), macrophages The transport mucociliary Green, 1974). bacterial studied the intercepte3 are alveolar Warshauer, and by removed physically P. haemol- presence the second hyperimmune These some may be two insight important 91 Materials Methods and Lambs All deprived, Mackay, lambs hysterectomy-derived, were were and McVittie reared (Hart, SPF conditions under Mellor, and colostrum- 1971). Vaccine vaccine used throughout was P. SSE, adsorbed onto Alhydrogel and emulsified as described in The Al oil, General Materials haemolytica type in mineral Methods. and Bacteria P. Materials haemolytica The in Methods and Preparation of pool Experiment type then first of 2 was transferred to and prepared lambs. weeks isinfected. . of in for pool life a room to in described as used antiserum two cleaned was antiserum colostrum-deprived the Al both experiments. 2 Experiment P. haemolytica from General type blood collected These lambs were under gnotobiotic which The had lambs been had Al used from reared for conditions, thoroughly no direct or 4 ý2 indirect contact with from acquiring protected intramuscularily first. Blood the and Al type for the lacked titre to inhibition old No antibodies in from in final the The type Al adapted to the injection *the clot, by antiserum of passing pool 1024, by detectable as blood results the filter. as haemolytica and sterilised P13 virus, assay intervals, collected after the after these was injections and haemagglu- microtitre plates 1975). A negative 6 week pool P. haemolytica to P. of filtration a 22 pm membrane lHA to basis 4 weeks 3rd and weekly at injected was r. --spectively separation by clarified 2nd the thus were P. haemolytica vaccine antibody serum After vaccine. tination ted the and the antibodies (Smith, IRA On the to prior it-through an of of Al taken were determined. was serum had titre serum type 2 months and samples preparation 2 weeks of 1 month given ional 3 occasions, on and animals unintezc. P. haemolytica infection. being conventional this control SPF lambs to serum P. pool was prepared haemo1ytica pool. of serum in type obtained a similar Al or from 4 to manner. P13 were detec- 93 1- Experiment Growth The aim phagocytosis in SPF lambs Design of P. in Lambs this Serum in demonstrate from cells wash the ne effect of type 2 were prior to ELISA the Al at vaccine from at intervals Antibodies in these technique as tyica vaccinated SC in all of 4 lambs brisket 3 weeks age. of samples were detailed in was class to measured by General Materials the at and sample IgG of 8 lambs 1,2,3, An additional slaughter. haemol of collected and vaccination 2 groups untreated. were samples to allocated P. haemolytica post-vaccination. the were 1 were weeks p. determ. Group Group of to lung the parameter. SPF lambs 2 ml with by haemolytica and to was in vitro serum and cells wash experiment vitro, Lambs each. lung in haemolytica experiment of Eight time of this upon vaccination of of presence P. 5 taken means of and Methods. Seven killed in to pairs nine weeks after (1 vaccinate vaccination, and 1 control) lambs were by injection IV 94 of 5 ml Ltd., May & Baker to lungs tipped fluid by sedimented were The 40 C. twice in more an Improved by exclusion majority types, cell grade each This for inoculum 6 hours in up screw was at tube a final prepared 37°C with and the was added The other although Culture present. tissue-culture Teddington, 2.1. each and 10 pl a 50 ml was of of concentration agitation. in estimated viability Table ml, washed counted were Ltd., 2.5 from cells AM, cells 20 minutes cytoplasm. (Sterilin in The g for plastic detailed culture was sterile the and 200 of were the covered jars. and the Lungs 0.1% funnel cells morphology caps as giving from lymphocytes as To each triplicate. suspension, blue had England) of volume set with Middlesex, The Haemocytometer, cells such tubes removed, was blood. gently, at the now with centrifuge centrifugation trypan the were mixtures kneaded were gelatin-Hanks. of trachea, was of by lungs filter sterile Neubauer of the a sterile supernate chest HBZS containing into into out mg/ml); removal the of sterile lungs The gauze. with pouring through removed, clamp at by (gelatin-Hanks) gelatin before clamped contamination avoid lavaged were trachea The England). Dagenham, the and opened sodium pentabarbitone (200 (Euthatal 10 total set up in P. haemolytica 5' ý NB culture, The The bacteria 10 5'9cfu/ml. incubated were Table Composition of Source Culture cultures of (16%) Serum 2.1 in Source 1 Experiment of lung wash (105.3/ml) A V V 8 C V C V - D C C E V C F C - V= vaccinate C= control cells 95 twice washed in at saline in saline an estimated These experiments lambs were 0.5 later, ice-cold the incubated experiments were g for cfu/ml. the as in bacteria of the supernate by the method of removed stop this the supernate. not made, and Misra 1.5 to added reaction, ml and Preliminary sedimented centrifugation did Miles, 120 minutes and and any but were 30,60 At rotation. with 4 minutes. that cells, wash of same day the 37°C and to 110 showed lung resuspended 107-108 of on at experiment, gelatin-Hanks, at count performed aliquots ml centrifuged the and concentration were were of start centrifugation killed. Cultures the by the alter viable and viable Ten-fold dilutions counts performed (1938). Irwin Results Antibody ni measured by the ELISA tech- ue The test, this the of sera for negative of P. haemolytica"as to the lambs P. haemolytica for vaccinated the 2 Group (unvaccinated) antibodies, duration lambs in are of the shown as measured experiment. in Fig. were 2.1. by The Specific titres Figure 2.1 Titres P. IgG of haemolytica ELISA in sera of measured by the type lambs, vaccinated to antibodies Al technique 0 Lamb No 10 0---® Lamb No 14 C-- Lamb ----10 o------fl No 22 Lamb No 32 600 / 30 14- O" Cu 0- 201 I. - 91 U a) 10( 1234567S9 Weeks after vaccination a 96a circulating IgG vaccination (lamb in 4 lambs all and rose Growth lung An analysis are from increase was less groups of cells than 1.5 in was the in the E) of postThe to up titres week presence of on each the results, 1enbs control the organisms by which contained D and P. haemolytica. in the regardless from the supernate these This effect whether vaccinated F), between (P1 d) U 0 N u 10 A N 0 0 a) rý k u ml rdl äl w 0 d ' Hr NIi1 r-1 d' 1 d' NI .N rQ rl A in N N U .N rl .,.I 0 r- NU 4.1 U1 d' 0 to O N H CT 0 d' d' CO NO d' d* Ln dý d' N d' d' Ln d' 4M d' 00 (Xi CO 0) 00 000 00 CO ND Ln d' 0 44 0 u id C) U 0 W W N Q) 0 r-i 9 0 Aj In a 4j OH z 0 ä n ui >lý H äý in ý 'ý ý T 0 z 0 in U) r0 0 4J 0 r-+ . -4 M . ONHII co M . to in . c%4 IH r-I 000N1 r4 r-d r-1 ON U) N 4) Ö ONrIi N H 1 N IH rd OD OOON r-i r-I Z N .r4 N id 1O N ý N N 4) AAAAA zzzzz d I ýtl II AAAAA zzzzz zý r4 Q 6 I w (Du N 00 U 0 H LAý Ný ý .. 4m r-- k-0 0 0 cO r 0 00 / L 00000 0o r` tDLý' 0 H H W 0 N U o Ow b 4a O r-I 0 41) r=: H, 3: Co 4 01 4' 0z II zz It 4c o l rn" . A ýn C4 v N (d J V O iflLl iI1 min 1II NO 11I u1cfd'OO inLo tod'n'l 1I r-1Mm 4) H' N U) O Iim1I ru 00 ic O zR .,4 ,I U) A öla N 1I NulH inLn -i-r-1 1 zÄ r-1 A Ri Ei r1 p0 u NU Ul 0 OH 44 co OD OD co OD 0)0)0)ß1ßl r-4 """". NO in cr'1mcýMCý qm 00""""" tN /o tn dý HH L. dý c4 4-I 0 0 rl """". n N U w N O P4 (L) >4 AAl E4 z Q in 0 Ln 0 to LÖ 0000 l0 . LO Ln in oi ö0 r-ro U, rq }.ý 0 fri co ýc . 1 I' I1I d' LO I 0 z ý N N $4 A V :3 O N 11t1 ro Htm11 KN d) '-I A H 4-1 U r-I U) :j0 0 H O H d' 00000 N/OLn4r4 d' d' d' d' LntnLO LO Ln 0otk Ln r-I 44 0 N U . 44 0 E Iz H >rt H O äj z 136 2- Experiment (types Vaccination A2 and Al, A6) It P. to necessary was haemo'lytica Al, be must A2, included sufficient to protect from The vaccine used in this experiment P. haemolytica types Al be or type A2. Vaccinated tica types Al, Design Forty of mice in each the were types two or one of for a vaccine with in use types would other types. SSE of contained and P haemolytica HKO of haemolY- P. with challenged A9. four A2 types (10 cfu/mouse). group each were and week Two weeks after the second unvaccinated groups Each each group haemolytica A6 cfu/mouse), sacrificed ml a two 6.6 Five 0.1 with four P. SC with vaccine, 40 control to of twice vaccinated 10 controls. and of cfu/mouse), 6.6 A9 (10 mice allocated randomly 10 vaccinates with A6, injections. these one A9 challenge P. haemolytica between injection, were A6 and were trivalent interval all challenge experiment of the A2, mice and if with and vaccine and in vaccination field, a trivalent A6 discover if the with mice haemolytica P. types with of controls at the consisting was namely, (105.0 and time mice of challenged Al (107.2 cfu/mouse), five of vaccinates challenge, or 137 the and mice remaining Viable were counts were 6 hours sacrificed performed liver on later. suspensions. Results The 4.3. Table Viable with challenged used in this mice with haemolytica the livers of of their were interval, mice challenged organisms mice P. with Six detected in challenged with indicating that content than 10 employed. cfu/ml, their the to A9, lower P. contrast, the livers type 6 hours groups after over Al those the same type challenge, of Al viable'organisms the A6 no homogenates of from vaccinated and type in counts of haemolytica limit challenged viable In liver of mice two livers the different hours the following in those and these haemolytica vaccinates 2'7 in 6 hours the mice haemolytica P. of significantly counts control vaccinated in controls. viable were in A2 and not dramatically. dropped in similar observed types respective time growth of types in summarised livers increased vaccinated infection after of are the four the of were P. in counts each Rates control experiment experiment challenge. of this of results counting any or was A6, less technique Text cut off in original 138 These protection A6, and This but show against challenge not suggests afford that results against that even same type. the possibility of p. haemolytica type against successfully A2. with P. with could types homologous 3 was afford haemolytica haemolytica P. Experiment the vaccine challenge HKO of protection, this designed immunising types A2 and type Al A9. A2 may not challenge to mice with investigate against 139 3- Experiment upon this from and Design of that A2 HKO in of a. type to made type lack of Experiment A2 protect A2, two 2 may have one made, of both using the and vaccine, The emulsified Vaccination Two groups with in were mineral of 0.1 oil varying 10 mice ml of the trivalent was as type each were P. in A2 HKO mal of ml dose ml dose of Alhydrogel described. HKO vaccinated haemol not possibilities mg HKO/0.1 onto the A2 vaccines previously amounts these type mg HKO/0.1 adsorbed either present these 0.227 0.682 other with in haemo? ytica contained antigens to haemolytica P. haemolytica P. due investigate To It parts. been antigen P. which to antigens insufficient monovalent several response between antigen. in performed Alternatively, protective occasions were haemolytica P. was the that or vaccine. and attempts competition vaccine. were haemolytica P. with }procedures experiment thought further, vaccination preparations. experiment vaccine, various with killed antigenic bea experiment This type of mice challenge living was of challenge in mice Effect tica SC on two type A2 HKO 140 the vaccines, Two weeks first. the these vaccine, each mice gastric at the time with type A2 in were 18 hours all tica mice the mice were haemolytica from each group five were killed for removed type A2 by prior and SC. type A2 killed were 6 hours enumeration and group were were washed type A2 group Eighteen gastric killed killed live giving of days in each, were mucin in of 20 mice at the 6 hours received treatment Two weeks IP with 6.9 cfu/mouse). later. of the received this group. time and containing 20 mice later, used saline suspension, of haemolytica inocula twice challenged (10 challenge P. The challenge. another 10 controls A2 in remainder to from mice One group 10 mice for repeated per protect saline. IP, inoculum type to P. haemolytica cfu, mice made NB cultures, in of ml was saline resuspended was and were haemolytica P. same control of immunisation Live An attempt 107.0 of after injection second cfu'P. mice 2 weeks given bacteria. viable 0.2 Five livers Their later. a group challenge, of the 106.7 with mucin. being after and IP challenged in b. injection second P. later, haemoly Five challenge, Their and livers 6 141 were removed c. Live and this in (10 A2 experiment, mice were at the time later; haernol in 0.5 ml later, All Five of and viäble counts mice the type survived and mice A2 dose. mice haemolytica cfu/mouse). challenge, type haemolytica P. these P. with tica a lethal with cfu/mouse). IP (10 mucin P. of with weeks challenged 6 hours killed type challenge 5.3 Three were killed to IP mucin challenge. were haemolyti_ca dose prior infected gastric gastric this of a sub-lethal were controls P. with part mucin, mice A2 in final with gastric Ten performed. mucin the infected in counts immunisation gastric In viable 10 type A2 per group rest were, performed type A2 were on livers. their Results Viable of mice in A2, the the given are A2 HKO vaccines, of haemolytica in mice or challenge Tables given live with 4.4-4.6. vaccinated p. The with p. in P. haumal and their haemolytica viable haeniol tica livers the treatments, vaccination after shown livers P. various controls, respective type of counts counts tica type type A2 either Key *** = Viable to Tables counts significantly after ** = Viable counts Viable * significantly after ND = Not challenge done. than 4.23 of vaccinates controls 6 hours (P In O +ý b 0 N O m r dý O N O $ .N s~ O U rd b U I r1 N 44 . r{ 41 4J ýo 0 u %D 43 r-1 P4 "rl U co 14 4-1 b b rZ H 4 O U 0 %D u) U a) A N0 U) HO O Ln "-4 .' OO u CC) O cN OO 10 a) U N U U0 r4 O 41 0 0 U N U .D co K u1 d' O . N 4) z 0 b 44 N U 44 0 a) 0 co ". -I Ua rý U b rl ni N U ro LI) u Lf) 9 aý N t0 P N 0 1.0 4 F: N z n ON 0 "r4 aN W Lo Ü r1 N 4C My d) N U) .. .ri U) co 9c tti 0 a) U) Q) 0 r"r4 01 r u ( Ln ui ri d) co C) v . rl 4J N r-l UO Ln M N u a) a 4-3 Lri G) r-4 A fU H U .. -1 4J f:4 C) Ln N 44 iv- G) U Um ný ? P4 5 u U rU H U0 NU Ll N Lf) LIl ., i Lo b. Lf a) a) Lr1 N 41 w 0 4-4 0 9) r-1 ro U Ci u1 U ((f lf) U1 rM 00 d' 'o L) l0 r-t 0 U N O w u U d' If) 0 U N r 0 u a; H "r Q -1 4i rt 0 . U b' 44 dc Q "r Q N 0U x 'N O0 rn 4) tr 4-3 4,4 f' (U d) E Cl) '--I r-i a) 0U P4 x w 0 .O ra C4 d) N" u E 0 N V C 0 ü 0 0 O NM aº 4 Q 4-I 0 U) d1 O 1 Co o N ao CO r4 (f) O 0 u u00 4 in co N . rq N u) N U u u00 C N U rn /o OD tN u r d' w cz ýCll %D in N u U C Ln . ri 0) u UA 0 y co GD .. l0 I OD d' r N n I fd H Ict U N N 4i b U d' r in 0'N N U rl .4J un w 0 U) O ý .4 43 0 0 rl 0 U C 01 u-) U0 U (U 9 m cc U . -1 b H u1 S 0i H . r4 r-I b 0 N 0 rl N Ri N4 G) U U W rd 0) U. O m ,)O N u) r+ 4 0 U W 4 0 O O 4 Co U 4) 4) 4-1 rd. I .,1 O O (1) b O C) mi u O 0 0) N U u W . ýC 0 Cl) ýo E 1 w"4 0 ri 0) N U A "Ü U Fi N O (d D 0 Si to N N m a, u .,ý O . Ln O +1 O u N co d) N 1~ . r4 U Ol U O rl to N ri co SI u1 rl N r-I to U c1 M O of N r-1 b O fd N OD N U N . U ro O r-I b a) rn 0 U 1{-4 0 ýr 00 b 4J 0 111 U O +1 If) i) Co r--1 r- 1 ri N Q) .--1 N a 4) in NI U U . r4 U N U H H p N %D O -F "u a" rI A ful E-1 HH ' O a, t HI "rl 4J Ü w 0 U N (V r-i 9 a) N p +1 H 0 N 4) ýo U U to CO rm 1.0 in . ur a) UN UN 1.0 4J r4 'o 4-3 rl O 1"+ 4.r I~ 0 u U I4 0) ft! UI m O N . r-I r-1 (a U () b u u (0 5 U M 4) is N H m O . O N +1 in N 0 U 43 O G) E is x w M 0 w 0 N b (9 4) ro a 4) tu u 4J äj 0 .I D N F. 4) ßl "rl äl b U) N W rd Q) N r-+ 0U I4 r-1 rc; O O1 a) U d) U F. v w rt W a) U . r{ r-4 O N 0 o -Ft O d) U 0 4-I 0 U) ki 0 0 l0 N CO d' 0 r1 4) r. 0 U W e O O O0 (V) . ri u 0 U CC) C,4 N W H 0 U 0 (W H "O b N 0 rl H fu N 41 U a) Itt H C) C) W rd rl 1 0 d' EH . rl M H N 4J a) U U N P4 Jv .lJ 0) (+) %D 00 N H U "UI ri OD n U H 0) a 4) t0 u U ro v tu U M Q) 0 0 ro E-', H O v rU Ea a w 0 to 43 w 0 0 U w r1 4i r-I cl id 0 4-) U U O U CO W N U ýO N ýt > 0 Co O M M N c ýO CO d' u) ro U %0 r4 M " Lo " OD 0 u aý 4.3 H ai H 4J LU U 0 U U 44 N ý b. H e-1 :3 Ic: 0 U In H O "D 44 it O M v o ft k7 N M rU 4J W N > äý N 4) O r-4 r-I 0 rz N N N H1 U N u "d F. H rS A ý u) N u F. 0 O i r4 0 N 43 rl 0 U O "ri 0 0 r4 Ea U) V W U U . 0 "x0 ýO d' . , . Ln ýo M 0 r-i H 4) r--1 r M O +1 H N N ".UI U O H P4 4J e. "kti -x -xOD . Ln 0 0) dt (rtf ri U Nr4F. H . 0 +t 4.) >1 > 01 a) rl Q) "ri U IQ al O rl . rq 9 d' E-i (: U d1 d` U M D ºn -K Lt) k r- 0 rl +t O A 4.) w 0 N I~ 0 U a) r-q rd 0) 4) U U .-i O 14 H M M r Ö N r 0 0 U A b 14 ti-I b 9: r-1 r+ .) a) W O ",i N (0 4 U 0 d' 0 +I a) U N I A A I 'r-I ' z z ' H E 44 m H 1 N 0 z z A I A z ' z z z I A z z z z z z z d) V) N r-1 N E m x U 4) ß Ow N d1 44 0 G) r-4 N 4) AH H 44 4c z 0 44 z z b d) N b U . ri 4-3 N p4 z z z z z z z z zr z ý Z z z z IC 03 44 0 z r+ ' ` IC ' ' H H Type of 2, monovalent haemolytica vaccines in Table Summary Type of of results 4.21 of P. haemolytica Experiments14-16 used for Iro T3 ý' y cI P) oýa'< öým N" co mr Comft :i N " rr n P) T4 T10 - ** T3 - T4 - - T1O - - *** challenge 144 SSE of both addition, P.3, type SSE types T4, of and against protected and P. types challenge haemolytica some protection afforded tica against mice protected In haemolytica P. of did the against haemolytica not protect P. haemolytica. mice the type Al A8, type P. types A2, All and type with T3 and haemolytica A5, challenge and homologous challenge with from A6, with haemolytica P. challenge P. Al, A7, type. A5 P. with vaccines haemoly- T4 vaccines type A9, TlO A12, T10. T3 homologous 145 17 - Experiment Vaccination HKO, A9 SSE and type of and 17 of possibility P. experiments Preliminary P. haemolytica HKO of some communication) personal to determine p. haemolytica type The vaccines coiiinations of their. P. of this two vaccine. Challenge mucin, cfu/mouse). in afforded (Gilmour, challenge designed was could SSE of combinations experiment two this be obtained with weeks in Groups occasions was A9 SSE and type with after of with P. the HKO, Materials 10 mice 0.1 ml haemolytica the various contained experiment detailed chapter. vaccinated (107.0 HKO. and a vaccine results haemolytica are SC on gastric SSE A9. used compositions section of experiment of Design this similar whether in A2 homologous and A9 that shown type against protection type had with composed vaccines using the challenge against haemolytica P. of combinations and A9, type haemolytica investigate to mice protecting haemolytica A9 designed was haemolytica P. P. with challenge type Experiment with mice second and Methods each were doses and of type vaccination A9. in 146 of 25 control five and from a group each the and retested was 25 of P. haemolytica mice challenge, 4.1) Table and mice was with enge (see 2 Vaccine type remaining (10 6.8 killed Chall- cfu/mouse), the at time 6 hours killed mice a group vaccinates. A9 were group using of later. Results These first the 6 hours part the of from turn in to heterogeneity. to appeared did the viable group controls counts of vaccinated by means to in vaccination In at mice of a t-test. found, were Some mice responded 4.23. and these partly groups others whereas not. Three of 6 hours killed to have 4.22 differences significant variance Tables each the with No statistically due in experiment, challenge after compared were shown are results the vaccine, 3 and vaccines for the after mice challenge whereas only 4 responded. repetition was chosen with larger numbers 4.23. There Table five of was vaccinated two mice For of mice. to appeared the this have the results variation responded groups reason, vaccination The considerable in 2 and vaccine with given 2 vaccine and are challenge given amongst in the 1 t'N U r+ OO CO D000 uN ri "ý1 ü ro r. CO UN ýM RM . tn ýn ýo Co cn O r-i..... o '"r1 1-0 d' 0-1 uý Ln O 0-1 aQ ar CO CO Co UN H O Ri l1 1i U 4a O U U 5 1 lle u UN (i '"ri QO ".. (J) O r-1 Co NO r'I c 1. un Ln U1 W CD eOO H Offl(H In ... S. . e0Loe m N O O co 00 O Ln Ln Co Co ý0 eCO ýo NNr 00 CO O 0" Ln O M H N ßl 0 Ln U ch r . LoinUn vU C0 o4. ..... -4NNýo NaouiNr eeM rl [N r4Q in O LO 0 m H 0 . 00 un """. Q1 de N wo LO r-+ N Q de CO e. cý N Q Ul d' CO in c`') Cl N N tN N " '-o c " Co 1 d. OO NOO0M in N H N ýn dý -,0 N . N (h 0 . Q f /.. to Q r0 NCl o' c) . O O . Q EnOD Coinin Co CO o ..... rN NNNr d) . r{ a U O 'ý N UIl N N d' ri 4 ä ro H 0 v 4J a ý! (D "r1 Co ON Od' OCQ. -+O ..... to in ýo lD 'o t() N U U N a U UN R U H N N d' CO O ß1 r-"1 a 00 is ".. ". Ln D Ln in in rn 4 in 0 Q 0 n CJCOQN u. .... / CD r- e d" -e GO o0 e r) . -1""1f" Co Om O 0 H O O CO c0 d' O cn CO d' tom 0 Cl t.n (L) s~ r1 cy 'r1 in OH " 10 in CO to CO 0) in 6 " " ýo O Co O H " " COs ""1 CO CO CO H d' D 0 " CO Q .ý b v 4i H N r4 .U OO CO00 CO 4 O Qrrnco C O !e un uI U U (d w 0 N s 0 U N . -1 ýd . r{ u) U . r{ E NN W 5z (d N H U) .-"t 0Ü OW u. O Q O YI COO eO Q Q ONO W `_ NNW Co CO N CO 0 Table Viable counts control mice of and P. Hours type haemolytica mice type 4.23 A9 vaccinated ("vaccine with A9 in P. livers haemolytica 2") after Controls challenge Vaccinates 5.74 5.74 5.81 5.93 6.00 5.90 5.90 5.93 5.95 5.98 x 5.84 5.93 SE 0.052 0.015 7.70 3.00 7.70 7.70 7.70 4.48 5.11 5.18 8.00 5.90 8.00 8.09 8.18 8.18 8.18 8.18 8.30 8.30 8.30 8.40 8.48 8.48 8.60 8.81 9.30 5.95 6.08 6.30 6.70 7.00 7.08 7.11 7.18 7.30 7.39 7.48 7.84 7.95 8.08 8.40 8.23 6.58*** 0 6 SE -0.090 0.302 of 147 viable counts nevertheless of the in the viable group vaccinated significantly livers the lower than of bacterial 6 hours the the vaccinated counts after controls in mice, the infection (P[...]... eleven bacteria Jones pneumonia an organism although appeared introduced was designate to cattle, had organism haemolytica P haemoiytica P of and group and III group Cross The of resembled 11 Group Jones' I and (The "typical" Pasteurella was has group it is but does not produce reactions of species as classified and and attacks certain were adopted are (Cowan P facultatively fermentation, 1974) Steel, and... considered only and point Vaccines still aimed in available that inadequate, vaccines this at are in that many the insufficient The general unsatisfactory pasteurellosis use immunising nature of vaccine field the by supported trials vaccine of This current against is possibility the of powers protect haemolytica disease of uptake or are P nevertheless outbreaks to latter disease of but the either or prevention... necropsied it cause, considerable in least its bacteria, lungs of the the organism is 1954) with a variety also of studied predominantly basis of important not cattle in of and Particular associated the certain types with is to is c and have organism pneumon' a and Europe been into subdivided and and or America biochemical the may sheep, pneumonia P haemolytica of the a septicaem North species from conditions... soluble immune in plasma T'e adsorbed homogeneous HuRBC, known was (1966) patterns Canada specific multocida Cameron haemoiytica of strains with in (1956) Carter P of which serologically of haemolytica strains shipping from of P subdivide agglutinated apparent from larger that a particular by 2 or numbers of 3 antisera strains a batch occasionally strain (1960) of P were of haemolytica (Biberstein, 1965)... I was the and the advice animal of photograI assistance Evans project by help of and Miss A technical many for C statistics, Skeoch J': Donachie Mr preparation Institute, and and technical Miss W to staff on for for thank the advice Inglis Mr and W Angus and for Aitken, D tests Mr K through- encouragement am endebted me on ELISA C I Dr Gilmour I and and advice due J M McLauchlan Easter and their and... pasteurellosis is associated parietal numbers spleen months of (Smith, contrast, age 21 100% of due type to Thompson has yet suddenly to been offered as be more of the winter The first all common in days The start of deaths The invasion have of Stamp, support Watt this to be and perhaps Thomlinson In several and may be be amongst The as cases found where of the in several best the to primary site has... sheep the 1978) factor Synergism been a P that (Sharp, haemolytica P of Rushton, theory rendering bacteria aerosol predisposing infection factors an infection a P13 virus of pasteurellosis pneumonic has establishment exposure to prior the to to exclude 26 a multiplicity of other factors 27* for Requirements and a suitable but able, P these are of due assess, to have attempts it tently successful the. .. trials vaccine against a limited cross-protection Field of haemolytica P against pasteurellosis only haemolytica, that of use, contain vaccines study vaccines their Despite for methods Commercial vaccine of prior serum to (Gilmour, test be may therefore Laboratory animals infection for either of animals which the has initial immunologi- 28 reproduction of pasteurellosis in reproduction of P haemolytica septicaemia... multocida Failure to is"oxidase-positive, it from most the milk haemolytica P characteristics, feature a useful of blue methylene these wbereas agar, not reduce to addition McConkey's on haemolytica P agar distinguishes which Enterobacteriaceae, which are oxidase- negative Satisfactory bacteria are fermentation often fermentation difficult media inconclusive strains biotypes, designated be associated... (1964) infections were reproduce Downey, Smith of Pulmonary llosis to achieved 1957; inoculation very factor induced was acidosis personal attempts Salisbury, bulin to the Pneumonia Early P acidosis sheep that by caused a predisposing prior These (Gilmour, sible of manipulation, disease, acidosis, may be a state P haemolytica the that that the prior to is little placenta the may affect disease there