COMPARATIVE TOXICITY 0F PASTEURELLA MULTOCIDA ENDOTOXINS FOR CHICKEN EMBRYOS Thesis for the Degree of M. S. MICHIGAN STATE. UNIVERSITY JAMES T. GARY, IR. 1971 rhtblf- m A.‘ 4M mun? Michigan State University I I A amine or T“ IIDAII £30k” -- BIIQII BINDERY INC. L1 QARY BINDERS I; " mum _-__ I I I ABSTRACT COMPARATIVE TOXICITY OF PASTEUFEZLA MULTOCIDA ENDOTOXINS FOR CHICKEN EMBRYOS By James T. Gary Jr. Eleven Pasteurella multocida strains selected to represent isolates from 5 different animal sources were extracted by 2 separate methods and their lipopolysaccharide antigens purified. Ultraviolet absorption at 280 and 260 mu indicated that all extraction products were essentially free of protein and nucleic acid contamination. The poorly-immunogenic nature of these preparations was indicated by their failure to produce a detectable antibody response in rabbits. Lethal doses of the purified lipopolysaccharides for young mice were high with MLD values greater 50 than 200 ug., and the localized Shwartzman reaction could be evoked in rabbits by only about half of the lipopolysaccharides tested. Intravenous inoculation of ll-day-old chicken embryos resulted in CELD50 determinations which differed lZS-fold for the 12 endotoxins examined. The endotoxin from a strain of P. multocida isolated from a dog was the least toxic of all preparations, and differed significantly from toxins extracted from strains representing the other animal species. No significant differences could be detected in the toxicity of lipo- polysaccharide components of strains from 3 of the 5 animal sources when examined by the chi-square (X2) contingency method of statistical James T. Gary Jr. analysis. Significant differences were found, however, between extracts from the avian PasteureZZa. The antigenic complexity of P. multocidb is discussed. COMPARATIVE TOXICITY OF PASTEURELLA.MULTOCIDA ENDOTOXINS FOR CHICKEN EMBRYOS BY James T. Gary Jr. A THESIS Submitted to Michigan State university in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department Microbiology and Public Health 1971 ACKNOWLEDGEMENTS I would like to sincerely thank Dr. G. R. Carter for his guidance and immeasurable assistance in the development and conduct of this study. Grateful acknowledgement is also made to Mr. A. Wayne Roberts for his assistance with many technical operations, and to Mr. Roberts, Mrs. Ann Steketee and Mrs. Dorothy Boettger for their individual efforts in helping to secure necessary supplies and other materials. Finally, I am deeply indebted to Dr. Charles W. thnson for his expert construction of the figures used in this thesis and to Harold and Pamela McAllister for the excellent photographic reproductions which made this work complete. 11 TABLE OF CONTENTS INTRODUCTION 0 O O O O O O O O 0‘ O O O O O O O O O O O O O O 0 REVIEW OF THE LITEM'NRE O O O O O O O O O O O O O O O O O O O O 0 Studies on the Chemical Nature and Physical Properties 0 f mdo to X1“ 0 O O O O O O O O I O O O O O O O O O O O O I General Characteristics of Somatic Antigens. . . . . Protein Component of the Complete Somatic Antigen. . Lipids Associated with the Complete Somatic Antigen. Polysaccharides Found in Somatic Antigens. . . . . . Dissociation of the Lipopolysaccharide Molecule. . . Biological Action of Endotoxins . . . . . . . . . . . . . In vivo Effects 0 O O O O O O O O O O O O O O O 0 O O Mechanisms of Endotoxin Action . . . . . . . . . . . Page UI ,Lp\o~qm hHHmomo.msouuomou ommowxo sous ano> oumuohnooumo as nauseous one neon oaon umnu oosonm uoumoaonu moo uo>wuamomo “meson on nouwm v mo>wumwozm 46 + + + + + +1 +I I +I +I +I +I +I +I +| +I I +I +I +I +I +I +I + one mqoalm owoanm «mmalm mnoalm ml<> main mwwlmo hamm HHQIA .. + + I + + + u .. + u a a 37m: ammo“: mumoaxo oumuufiz mom unsung HoooH omouosm Housman: omouanz amouoma omoooaw mmxnooonz "samuum aoum mau< so nusouo henna menu a“ vow: moamuum unwooeNxs .m on» mo mooauomou Hmoflsonooam .H canny 47 Table 2. Animal source, associated disease and serotype of the various P. multocida strains Strain Source. , .Associated disease Serotypea ATR-751 Primate Peritonitis Not known L-6ll Canine Not known Not known ET 3397 Swine Pneumonia l:A : 63-875 Avianb Fowl cholera Not-known B-19 Avian Fowl cholera Not known VA-3 Avian Fowl cholera 5:A i» P-1059 Avian. Fowl cholera 8:A P-1234 Bovine Hemorrhagic septicemia -:E B-1046 Bovine Not known B-1048 Bovine Not known 656 Bison Hemorrhagic septicemia 6:B aBased on the serological groupings proposed by Namioka.and Murata (81). 48 studies along this line are pursued. The present study, however, was intentionally designed to examine only the general relatedness between endotoxins from strains of different animal origin (see Discussion). Ultraviolet Absorption The ultraviolet absorption of purified LPS preparations was examined by scanning wavelengths between 230 mu and 300 mu. Absorbancies were recorded onto graphs and the values noted at 280 my and 260 mu were used to determine concentrations of protein and nucleic acid, respectively. E The results are shown in Table 3. It will be noted that for both methods employed the rigorous purification-process resulted in products containing negligible amounts of these substances. With only a few exceptions there were no peaks of absorbance recorded over the wavelengths scanned. Removal of Capsules Mucoid strains of P. multooida were subcultured up to 10 times on blood or tryptose agar in an effort to obtain nonmucoid variants. Colonies demonstrating slight, or no, mucoidness were selected and these ‘were used for the extraction of LPS. All strains were washed for a con- siderable length of time in saline before extraction and ethanol frac- tionation was carried out-later in the process. Although absolute removal of nonendotoxic polysaccharides was not considered likely, it was anticipated that only trace amounts would remain in the purified extract 0 Immunization Results One characteristic of lipopolysaccharides which has been reported many times in the past is the poor immunogenicity of these substances. Specific antibodies have rarely been demonstrated after immunization with 49 Table 3. Protein and nucleic acid content in extracted lipopolysaccharides -— j r 1 Extract Method of Absorbance Percent contaminantse (gghgg From? Extraction 280 nm. 260 nm. Protein Nucleic acid AIR-751 Phenoldwaterc 0.075 0.075 0.060 0.002 L-6ll Phenoldwater 0.014 0.020 0.010 0.0006 F77 3397 Phenol-water 0.030 0.017 0.040 0.00 I 63-875 Phenol-water 0.011 0.012 0.003 0.0004 B-l9 Phenoldwater 0.017' 0.008 0.020 0.00 VA-3 TCA-Phenold 0.032 0.030 0.025 0.001 I p—1059 TCArPhenol 0.014 0.00 0.020 0.00 '7‘ P-1234 TCA-Phenol 0.008 0.010 0.003 0.0004 B-1046 TCA-Phenol 0.012 0.012 0.003 0.0004 B-1048 Phenolrwater 0.003 0.004 0.001 0.0001 656 TCA-Phenol 0.010 0.00 0.010 0.00 0126:Bl6b' TCArPhenol 0.051 0.083 0.002 0.020 8Strain of P. multoeida from which the purified LPS was obtained. b cModification of the Westphal extraction procedure (see Materials E. coli. and Methods). -dMethod of O'Neill and Todd (see Materials and Methods). eInterpreted from a nomograph prepared by E. Adams (see Materials and Methods). 50 protein-free LPS. With these reports in mind, an attempt was made to elicit the production of antibodies against the materials extracted for the present study.“ Two rabbits were immunized with each of 6 different extracts as reported in Materials and Methods. No precipitates were formed in immunodiffusion plates between any.of the purified endotoxins and their "homologous" serum. These results suggest that there was insufficient protein complexed.with the molecules to evoke active synthesis of specific .mr antibody. The possibility cannot be discounted that antibodies may have been produced but in quantities too small to be detected by the systems employed. In any event, the absence of a detectable, specific immune response supports a poorly-immunogenic, protein-free, lipopolysaccharide 1.:- as! character for the preparations extracted. Immunodiffusion Studies The reactions noted in.gel between the extracts and antisera pree pared against whole bacterial cells-provide even greater support for the LPS nature of these substances. It can be seen in Table 4 that several of the purified extracts formed precipitates with one or more.of the antidwhole cell sera. Most of the lines of precipitation were located in very close proximity to the antigen well corresponding with the normal location of precipitates formed by lipopolysaccharides. Strains P-1059 and 3397 reacted with several of the antisera suggesting that common somatic antigens, or antigenic determinants, are shared by at least some of the members of this species. Another interesting possibility results from the finding of Prince and Smith (107) that "a" antigens present in the capsules of bovine and avian strains which they examined are extremely difficult to completely separate from the cell wall. These components were found to be antigenically similar to somatic Y antigens and to 51 Table 4. Reaction of purified lipopolysaccharides with antidwhole cell P. multocide serum in immunodiffusion plates Antigen Antisera Precipitate (LPS from) Reacted with Proximity to.antigen well Comments P-1059 P-1059, VA-3, Very close; bordering anti- Single distinct 989, CD-90A gen well with some sera line 3397 P-1059, VA-3, Bordering or very close Single band; not 989, CD-90A very distinct B-1048 989 Approx. 1/4 distance toward very_broad band. antiserum well Appeared to rep- resent a single antigen CD-90A CD-90A Bordering Single line; small but dis- tinct L-6ll VA-3 Close Board; indis~ tinct band 656 989 Very close Single small indistinct band 52 possess type-specific antigenic determinants in addition to some nontype- specific determinants which were shared by.noncapsular components of other types . Yield of LPS The extraction procedures employed by this worker yielded very low amounts of purified LPS, with the average range lying between 2 and 4 mg. of LPS per 500 ml. of cells extracted. A relatively low percentage yield was anticipated since essentially complete removal of all contaminants was the purpose of the adopted methods. However, it was expected that somewhat more than 4 mg. of purified toxin would be obtained from the cells grown in 500 ml. of medium.‘ Mouse LethalityATests Injection of mice with the quantities of LPS already reported (Materials and Methods) resulted in an insufficient number of deaths to obtain LD50 values. Less than 50% of the mice were killed when 200 ug. of endotoxin was injected. From these observations it became evident that the quantities of LPS which would be required to adequately determine and compare lethalities of these substances for mice would severely limit in number other experiments more closely related to the proposed purpose of this study. Hence, investigations along this line were discontinued, and no data regarding mouse lethality is tabulated in this report. Induction of Shwartzman Reactions Table 5 shows the results of an attempt to induce localized Shwartzman reactions in rabbits. Three strains failed to produce noticeable skin reactions. There appear to be several possibilities that might explain the inconsistent results; however, since the number of animals available rI 53 Table 5. Localized Shwartzman reactions resulting from P. multocida lipopolysaccharide injection into rabbits *- u—v" - —— Shwartzman Extract from Reaction (cm. ‘) Comments P-1059 3.60 Sizeable necrotic area, charac- :HT‘T teristic reaction a I B—1048 1.65 Necrotic destruction slight ' P-1234 1.00 Small area of hemorrhage I I 656 N.R..b Several petechiae, no basis for E measurement a 3397 1.60 Marked hemorrhagic necrosis, very characteristic reaction 63-875 N.R. L-6ll N.R. TArea - in sq. cm. - calculated as the product of 2 diameters ‘measured at right angles. bNo reaction. 54 for testing was limited and only one was injected with each preparation, any.attempt to explain such inadequate data would be speculatory. In addition to the possibility that insufficient LPS to elicit a reaction may have been injected, one might also consider such proposed influences as prior exposure to related organisms (123) or to a protein component (31), and the induction of hypersensitivity (61). Toxicity of P. multooida for Chicken Embryos In Table 6, data pertaining to the chicken embryo inoculation experi- ments, which constitute the foundation of this thesis, is summarized and its significance indicated. Fifty percent lethal doses differed 125-fold between the lowest and highest values obtained. It will be observed, however, that in every experiment recorded, the results were significantly different from.any result which would be expected due to random chance occurrence.‘ (In all but one experiment P was less than 0.001.) No significant differences were detectable between the toxicities observed 'when 2 or more experiments were performed using the same extract. These evaluations confirm the reliability of the data obtained and the reproduci- bility of results. Statistical Analysis of Toxicities Since the main purpose of this investigation was to comparethe toxicity of endotoxins on the basis of their animal source, at least 2 strains were selected which.were isolated from each animal species repre- sented in Table 6. The first extractions, however, were made on the growth from 250 ml. of broth, and because of the low endotoxin yield mentioned above, many of the products were not of sufficient quantity to 'be tested. In several instances the total yield was less than 1.0 mg. of purified LPS and this was not thought to be an adequate amount to work 55 .m.z Heo.o v.m o m _ oeoo.o m\d. 0H\n ¢H\¢H 0H\0H ono souwm Hoo.o I m H n «moo.o 0H\m m\e a\m w\w ono Hoo.o v a H a oHoo.o HH\H ~H\NH a\m ~H\~H «manna .m.z Hoo.o v m o o oooo.o N\o m\H o\o oa\oa «mmdlm Hoo.o v m 0 ma _Hmoo.o NH\H HH\~ HH\HH HH\HH emNHIm .m.z Hoo.o v m o o nmoo.o nH\o nH\AH nH\¢H mawwa oqoalm .m.z Hoo.o v m o o smoo.o naxo ma\m ¢H\¢H naxna mooalm osfi>om Hoo.o v m o o «moo o nNG. o\w n\n I oooHIm .m.z o o omoo.o n\H n\m «\e I oooalm Hoo.o v m N CH omooo.o 0H\N w\w w\w m\m oqoalm Hoo.o v m o s mmooo.o ~H\~ NWVNH HHNHH w\m amoHIA .m.z Hoo.o v m o n muoo.o ~H\o NH\OH MH\MH mH\ma mnoanm Hoo.o v m o o owooo.o «\m ma\oH wa\wa m\m mnoalm Hoo.o v m o o omoo.o HH\A NH\w OH\OH ~H\~H onoalm I Hoo.o v m o HH «moo.o w\o NH\H owww aflwoa malm Hoo.o v.m o o mooo.o nxa n\a o\o n\n malm smH>< I maoo.o v m o u once o m\o m\m oH\n a\w, ml<> o n «moo.o o\o nxm n\~ n\n mum» w.m.z Hoo.o v m 0 ea onNo.o na\d. nH\o nH\o «H\ma nnwtmo Hoo.o v m o n omno.o NH\o oa\o HH\m HH\m nnwumo omooeouommwn omen uoouoonsH mm.w:v A.wnv A.wnv A.wnv A.wnv Bonn mouoom mo oosooamwewam maonusoo ammo mooo.o woo.o No.0 oa.o uomnuxm Hmsfin< moonwasoosw .oaNooHo .osumohunsm mesouw canoes noooouommao no cosmofimwnwam one momoo apnoea cosmos .ooumaooosw momunsm "some soaaasoo .o mHan .wmummnou mum madmawuoaxo mm muomuuxm mo huHHmnumH a“ acaumfium> mmumoflvaa qaaaoo .uamuuwwawam nozm . .muamnoIou 05v vmuomnxo mp uddo3 noa:3.mmosu scum muasmmu vm>uomno mo ounmummmaw mo monmuwmwawfim msu muammoummu naaaoo mwnu nu damn .munmafiummxm N «o coaumafinaoum .wNoo .mw .Amwonumz van mHmHuoumz.momv wonuua muamwafluaoo N man up vmumaauawo N v .oawamm vanadfiamauom .Ha mo.o saws kumasooaHu .Acv “madame cam Hoaafiz mo uosuaa nanoum wsu mp vmumaflummn .umnasa was» a“ vmvaauda uoa.mum ammo mm3_mmmnuuoamn :ofi33.aa moaunamm 56 .m.z Hoo.o v m o ,5 mqoo.o o\o "\w INN“ mxm oflmuomflo Hoo.o v m o o quoo.o HH\~ Na\m ~H\HH ~H\HH «camuomflo I .Hoo.o v m o w mmoo o ma\o ma\~ OH\OH mum annuma< mumafium o o mmoo.o I ~\q o\o o\o HmnumH< .m.z .Hoo.o v m N «a oNoH.o owwc ¢H\o oa\~ ¢H\m Haouq magnum no.0 m o q Imnmo.o o\o “\o OH\H m\u4 dawn; Hoo.o m H HH «moo.o HH\o Ha\m oa\oa mum uamm .m.z Hoo.o m o o «moo.o a\H .¢\m oH\m ,oa\oa Rama mafiam o m oqoo.o «\o «\N n\m o\o "mam Hoo.o m o q Hmoo.o «\o «\N m\¢ n\m “mmm vmmoamummman vman cumuoufiaH mm.wnv A.m:v A.wnv A.wnv A.wnv Scum monsom mo muamofimfiawfim maouuaoo name mooo.o «oo.o No.o oH.o pomuuxm Hmafla< «@9630 on.“ . oahuoflu . on" manna—am A.u.uaoov o manna 57 with and obtain results which could be considered reliable. Because of the time and labor required to carry out an extraction as outlined in this report, it was not practicable (within the limitations of this project) to repeat this process for the purpose of increasing the effect- iveness of comparisons. An adequate amount of LPS was obtained from several strains of bovine and avian origin. Statistical analysis showed that none of the bovine strains differed significantly with respect to the toxicity of their somatic antigens for chicken embryos. Among the avian strains, however, highly significant differences were observed. Table 7 shows the results of statistical comparison of lipopolysaccharides from avian strains. The extract from strain P-1059 differed significantly from those of other avian strains, while a closer relationship was noted between the rest. The method of extraction apparently made little difference. In fact, a comparison of the mean toxicities of extracts obtained by the different methods showed that the TCArphenol extracted LPS was only slightly more toxic than that extracted by the phenol-water procedure. Since strain P-1059 represents the most frequently isolated serotype found among avian strains, the possibility that its greater toxicity might reflect its more frequent. occurrence in nature was considered. Plate agglutination experiments showed that cells of strain 63-875 (the least toxic avian strain) were agglutinated by antiserum against strain P-1059. Although a reaction with only the capsular antigens of this strain was not excluded as an explana- tion, it is obvious that until the immunotype of 63-875 is completely determined, no valid correlation can be made between the prevalence of strain P-1059 (serotype 8:A) and the toxicity of its LPS. In Table 8 are shown the results of statistical comparisons between lipopolysaccharides from strains having different animal origins. Several Table 7. Statistical comparison of differences in lethality among lipopolysaccharides extracted from avian P. multocida Extracts Compared X2a Significance of Difference 63-875 vs. 63-875 0.31 N.s.c 63-875b vs. VA-3 4.11 N.S. 63-875 vs. B-19 7.49 P = 0.05 63-875 vs. P-1059 63.13 P << 0.001 VA-3 vs. 13-19 0.45 N.S. VA-3 vs. P-1059 21.94 P < 0.001 B-l9 vs. P-1059 16.16 P < 0.01 p-1059 vs. P-1059 3.83 N.S. All extracts 68.78d P << 0.001 aTest statistic; the Brandt-Snedecor formula was applied in order to partition off total Xz's into individual comparisons (4). be no significant difference existed between individual toxici- ties of an extract, all experiments done with that extract were combined and comparisons made. cNot significant. dTotal X2. 59 Table 8.‘ Statistical.comparison.of differences in the toxicity of puri- fied P. muZtocidb lipopolysaccharides for chicken embryos, based on the animal source of the organism Extracts Compared (animal source) Xzb Significance of Difference Total avian vs. total bovinea 8.67 P < 0.01 Avian vs. swine 64.42 P << 0.001 Avian vs. canine 111.16 P << 0.001 Avian vs. primate 66.42 P << 0.001’ Avian vs. E. coli 67.65 P << 0.001 Bovine vs. swine 2.61 N.S.c Bovine vs. canine 76.21 P << 0.001 Bovine vs. primate 0.31 N.S. Bovine vs. E. aoli 0.27 N.S. Swine vs. canine 37.65 P < 0.001 Swine vs. primate 0.84 N.S. Swine vs. E. coli 1.36 N.S. Canine vs. primate 39.00 P < 0.001 Canine vs. E. 0016 44.35 P < 0.001 Primate vs. E. aoli 0 N.S. aAll experiments with strains having the same animal source were pooled for the comparisons reported.‘ bTest statistic; see a under Table 7. cNot significant. 60 highly significant differences can be observed. Comparisons reported between avian and bovine strains were made by pooling the data from all experiments with each strain and testing it as a single observation. Although the resultant X2 is highly significant its size was decreased by pooling the data among the avian strains. P-1059 was significantly more toxic than all of the bovine strains which were, in turn, more toxic than the other avian strains. Although the total X2 resulting when P. muZtocida from.other animal hosts were evaluated against the entire avian group was very large for each case, individual comparisons showed: (a) P-1059 LPS was significantly more toxic but that from 63-875 less toxic than LPS from 3397, while LPS from VA-3 and B-l9 did not differ significantly from 3397; (b) LPS from the canine strain (L-6ll) was significantly less toxic than those from all avian Pasteunella except 63-875; (c) endotoxin from 2 avian Pasteurella was less toxic than the material isolated from a primate strain, while for 2 other strains there was no significant difference; (d) the toxicity of LPS from the E. coli strain was identical to that from.the primate strain and identical results were obtained when this preparation.was evaluated against extracts from avian strains. Two P. multocida strains of canine origin were selected for use in this study. Both produced the so—called "blue" colonial variants, with no demonstrable capsule. After extraction only a minute amount of LPS 'was recovered from one of the strains. A group of embryos was inoculated with some of this material in an attempt to obtain results referable to those recorded for the second LPS (L-6ll). However, the CELD50 for this experiment was much greater than 0.10 ug., so the data were discarded. Lipopolysaccharide from the canine strain L-6ll was the least toxic ‘material examined in this study. Its toxicity was of a significantly 61 lower order than the toxicity of any other preparation except 63-875. Bovine, swine, primate strains and E. coli did not differ significantly from one another. 7" The experiments summarized in Table 9 were added because of their potential importance, after the main studies had been completed. The cells were not treated extensively and it is therefore not possible to declare that all capsular substance was removed. No correlation of the data obtained from these experiments could be made with the data from chicken embryo inoculations. 62 Table 9. Toxicity of whole P. multocida cells for chicken embryos No. of embryos dyingZNo. inoculated_per dilution CELD50 a Strain Undiluted 1:10 1:50 1:250 1:1250 1:6250 Control (cells/ml.) 3397 - - 9/9 9/9 5/11 1/7 0/6 5.3 x 103 P-1059 - 5/5 10/10 6/11 2/10 1/10 0/5 9.0 x 103 p-1234 10/10 10/11 5/10 0/11 0/7 - 0/6 4.3 x 104 656 - 9/9 11/11 8/10 2/10 0/9 0/11 8.8 x 103 ATR-751 - 10/10 12/12 1/11 0/11 0/11 0/6 5.8 x 104 aEstimated by the probit method of Miller and Tainter (4). DISCUSSION This study was aimed at utilizing the acute sensitivity to endotoxin of intravenously injected chicken embryos to detect differences, if any, between P. multocida strains isolated from different animal sources. A 125-fo1d variation in the estimated CELD50 was noted and some strains differed extensively in their toxic properties from others. However, for several groups, separated on the basis of their anima1.origin, no statistically significant differences could be detected. A considerable body of evidence points to the difficulty in determin— ing distinct differences between the members of the species investigated here: MacLennan and Rondle (71) extracted a lipopolysaccharide.with which they were able to demonstrate a single antigenic component. Only one line of precipitation was formed in gel when the LPS was reacted against its homologous antiserum; however, it was necessary to remove or reference (by the formation of lines of identity) nontype-specific antigens to identify this component in whole cells. Bain and Knox (3) commented that the antigenic complexity of P. multocida has always been underlined by the numerous cross reactions which may be noted in all attempts to type the organism. Carter and Bain (20) asserted that the difficulty in evolving a satisfactory serological classification of P. multocida was mainly due to the remarkable capacity for variation, the close antigenic relationship of strains and the frequent occurrence of strains among animals commonly employed for the production of immune sera (20). Namioka and Murata (83) stated that the pathogenicity of 63 64 P. multocida varies with each culture. These workers also implied that the capsule of this organism has an influence upon its virulence and they were able to divide its 0 antigens into 2 components, common and specific antigens. Heddleston (41) reported a high degree of cross-reaction between "particulate" antigens isolated from a virulent bovine and an avirulent avian strain. He postulated that the cross-reactions might result from the presence of antibodies directed against a common component which is present in both preparations. A capsular component designated "a-antigen" was found by Prince and Smith to possess some antigenic similarities to the somatic, or "y" antigen (107). The capsular a antigen was further stated to be a complex which was type-specific in Carter's type B and E strains but also in possession of some nontype-specific antigenic determin- ants which were shared with noncapsular components of other types. These researchers successfully induced cross-stimulation of capsular antibody production within bovine and avian strains and between the two. Thus, it was suggested that the a-complex probably contains a common protective protein fraction whose ability to protect is dependent upon its availa- bility in individual strains. The variety of observations (and speculations) summarized above make specific proposals on the basis of the incomplete data presented in this report unsupportable. The added support which would be provided by data relating to several strains from each animal host becomes obvious when the lack of relatedness found within the avian strains is considered. 'Whether incomplete removal of capsular substance may account for a portion of the differences cannot be definitely stated; however, the author is inclined to discount this possibility, due to the close morphological similarity of all capsulated strains at the start of the extraction pro— cess. It is interesting to note that strains P-1059 and 3397 cross-reacted 65 with several antisera in the immunodiffusion tests. These results have been observed many times with whole cells and LPS from these strains, sometimes after 3 or more rigorous saline washes had been performed. There is no "enlightened" explanation for this at the present time; however, one can perceive the extreme difficulty which will be encountered in trying to apply to such strains criteria which would indicate.a spe— cific animal-host relatedness. Further tests using more strains from each animal source would be necessary before a meaningful evaluation could be made. As indicated above, there is some.evidence that several somatic or. capsular fractions (e.g., the a, B and y antigens of Prince and Smith) may participate to a varying extent in the toxicity of P. multocido. This suggests a need to isolate and purify the various surface and external components and to test each for toxicity. Further characterization of these fractions, once purified, would also be of great value. One other procedure which would appear to be in order if subsequent studies are conducted is the testing of embryos for antibodies against P. multocida. Such tests were not performed during these studies; however, the presence of P. multocida antibody, no matter how unlikely, should be determined because of the information a positive result could later provide in respect to the comparative toxicities of different serotypes. SUMMARY In summary, lipopolysaccharides were extracted and purified by 2 different procedures. Eleven strains of PasteureZZa muZtocido were used for the study. The antigens were not immunogenic in rabbits and only about half of those tested evoked a localized Shwartzman reaction. Preparations containing up to 200 ug. of lipopolysaccharide demonstrated little toxicity for young mice. A 125-fo1d difference in the lethality of toxic extracts injected intravenously into ll-day-old chicken embryos was found. Although some significant differences existed, no definite correlation could be made between toxicity for chicken embryos and animal source or prevalence of the limited number of strains examined in this study. 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