A SEROLOGICAL STUDY OF STRAINS 0F PASTEURELLA MULTOCEDA ISOLATED FROM PRlMATES

Transcription

1 A SEROLOGICAL STUDY OF STRAINS 0F PASTEURELLA MULTOCEDA ISOLATED FROM PRlMATES Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY NINA C. RUNFOLA 1970

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3 ABSTRACT A SEROLOGICAL STUDY OF STRAINS OF PASTEURELLA MULTOCIDA ISOLATED FROM PRIMATES BY Nina C. Runfola An attempt has been made to differentiate twentytwo primate strains of Pasteurella multocida into the known serotypes of this species. a. multocida has been separated into serological groups on the basis of capsular antigens (14, 29, 46) and into different types on the basis of somatic antigens (37). In this present work, differentiation of capsular groups has been attempted by indirect hemagglutination tests. Somatic antigens have distinguished by gel diffusion analysis of lipopolysaccharide obtained by phenol-water extraction of cells. Since somatic antigens are part of an endotoxin or lipopolysaccharide complex, chicken embryo lethality tests have been used to determine the endotoxicity of the preparations. The use of the fluorescent antibody technique was attempted in an effort to detect capsular antigens. However, type specific fluorescein conjugates of antisera could not be obtained and this technique was observed to be effective only for species identification.

4 A SEROLOGICAL STUDY OF STRAINS OF PASTEURELLA MULTOCIDA ISOLATED FROM PRIMATES BY.r 2/ 1 Nina CiyRunfola A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1970

5 TABLE OF CONTENTS Page LIST OF TABLES O C O O O O O O O O O O O O 0 iii LIST OF FIGURES iv INTRODUCTION REVIEW OF THE LITERATURE Early Studies Capsular Studies: Colonial Variation. Capsular Studies: Nature of Capsular Antigens Capsular Studies: Classificatio Based on Capsular Antigens Somatic Antigens: Isolation and Characteristics Somatic Antigens: Classification Based on O Antigens Relationship with Other Gram-negative Organisms MATERIALS AND METHODS Cultures Media Preparation of Antisera Indirect Hemagglutination Test..... Fluorescent Antibody Technique..... Capsule Stains Gel Diffusion Technique RESULTS DISCUSSION BIBLIOGRAPHY O O O O O O O O O O O C O O O 0 42 ii

6 LIST OF TABLES Table Page l. ORIGIN, ASSOCIATED DISEASE AND COLONIAL FEATURES OF THE PRIMATE CULTURES RESULTS OF CAPSULE STAINS OF PRIMATE CULTURES C O O O O O O C O O O O I O O O 3. RESULTS OF PRELIMINARY RAPID SLIDE AGGLUTINATION TESTS OF SALINE EXTRACTS. 4. HEMAGGLUTINATION TESTS OF SALINE EXTRACTS EMPLOYING VARIOUS GROUP A SERA PRECIPITATION REACTIONS OF SOMATIC ANTIGENS OF THE PRIMATE CULTURES LIPOPOLYSACCHARIDE YIELDS OF THE PRIMATE CULTURES I O O O C O O O O O O O O O O O 7. CHICKEN EMBRYO LETHALITY TESTS OF LIPOPOLYSACCHARIDE PREPARATIONS OF THE PRIMATE CULTURES SUMMARY OF RESULTS OF SEROLOGICAL TESTS iii

7 LIST OF FIGURES Figure Page 1. GENERAL SCHEME OF ENDOTOXIN STRUCTURE.. 12 iv

8 INTRODUCTION Throughout the years many proposals for a serological classification of Pasteurella multocida have been suggested. The significance of such a classification lies in the necessity for effective vaccines for protection against several diseases of domestic animals. It has been found that certain serotypes within this species are responsible for certain diseases. Early workers used host specificity as a criterion of classification, while others utilized fermentation tests. The presence of different specific capsular antigens made possible a classification into capsular groups. More recently, the differentiation of specific somatic antigens has allowed a classification based on both capsular and somatic antigens. Examinations of strains of P. multocida isolated from many domestic animals have previously been made. However, only very few monkey strains have been studied The strains studied in this present work were obtained from the National Center for Primate Biology at Davis, California, and most were isolated from monkeys with clinical symptoms of disease. 1

9 At present, four capsular groups are recognized within this species, and are designated by the letters A, B, D and E (14). The method of choice in the separation of these groups is indirect hemagglutination. By this method, erythrocytes are treated with capsular extracts of bacterial cells. It is believed that some of the underlying lipopolysaccharide is extracted with the capsule and that this lip0polysaccharide adsorbs to the erythrocyte, with a resulting exposure of capsular antigens on the surface (2). In the presence of specific antiserum, hemagglutination then occurs. Eleven somatic antigenic types have been ident ified by Namioka and Bruner (34) by means of agglutination tests, and have been designated by the arabic numerals 1 through 11. These workers have used both capsular and somatic designations in the serological classification of the species. It was found that a single somatic type might be associated with several capsular groups. In this present work, somatic antigens have been extracted by phenol-water treatment of cells, followed by ethanol precipitation. Merthiolated saline solutions of the lipopolysaccharide were placed in gel diffusion plates for antigenic analysis. Initial studies of the reactions in gel diffusion plates indicated the presence of a multiple antigen-antibody system, and for this reason,

10 capsular extractions were performed previous to phenol water extraction in order to remove capsular antigens. The endotoxicity of lip0polysaccharide preparations was determined by a study of the biological activity, since no single chemical criterion of endotoxin purity has been described in the literature. Chicken embryo lethality tests were used as indicators of biological activity.

11 REVIEW OF THE LITERATURE Early Studies Pasteurella multocida is the Species name for a group of Pasteurellae characterized by an absence of hemolysis, the production of oxidase and indol, the absence of urease, and a failure to grow on Mac Conkey's medium (17). This group of bacteria includes the causative agents of hemorrhagic septicemia in cattle, fowl cholera and swine pneumonia, and has been implicated as a secondary invader in various diseases (16). The species name Pasteurella multocida was not used until 1939 (47). Initially a zoological classification based on host specificity was employed (28). The organisms were named according to the host species from which they were isolated--hence the names P. boviseptica, P. aviseptica, P. suiseptica and so forth. Inherent in this system of classification were several disadvantages. In the first place, the organisms responsible for a single type of disease were identified by different names. Secondly, organisms grouped under one species name might cause several different types of disease. All of this led to much confusion.

12 Several workers, however, recoqnized the possibility of a serological classification. Among the first of these was Cornelius in 1929 (22). Employing an agglutinin-absorption test, he separated twenty-six strains into four groups. He also observed a lack of correlation between serological group and animal origin of his bacterial strains. Observing that some of Cornelius' strains tended to be inagglutinable, Yusef (50), in 1935, utilized a precipitin test, in which fourteen of his twenty-one strains fell into three serological groups. By agglutination tests, Rosenbusch and Merchant (47), in 1939, observed three serological groups, which also exhibited distinct differences in their fermentation of xylose, arabinose and dulcitol. They suggested that a serological classification replace the earlier zoological classifica tion and that the name 3. multocida replace the host Species names. They did observe that all of their avian strains fell into their Group I, although their Group II comprised strains of all origins except avian. Little and Lyon (29) demonstrated the existence of three serolog ical groups by a rapid slide agglutination test. Their passive immunization tests indicated that monovalent serums protected mice against organisms of the homologous, but not heterologous, type. In 1947, Roberts (46) found four immunological groups by means of cross protection tests

13 in mice. He did, however, observe strains which did not fall into any of these groups and therefore suggested that other serotypes might exist. Capsular Studies: Colonial Variation By a study of colonial morphology, the existence of several colonial variants was demonstrated within this species. Although terminology differs throughout the literature, three main variants have been described: iridescent (smooth), blue (rough) and mucoid (7, 18, 20). The most common methods of distinguishing these variants are observation through obliquely transmitted light and an acriflavine test (7). By means of obliquely transmitted light, smooth colonies appear iridescent, usually of a somewhat greenish nature, mucoid colonies exhibit a slight reddish iridescence, and rough colonies appear noniridescent (7). Mucoid colonies are usually the largest in size and are mucoid or slimy in appearance. Smooth and rough colonies are generally smaller in size and lack a slimelayer. In acriflavine, smooth colonies remain in suspension, rough colonies clump and mucoid cells form slimy precipitates (7). Capsular polysaccharides from iridescent variants differ both biologically and chemically from those of mucoid variants. Those isolated from iridescent variants are immunogenic and serologically

14 active, while those from mucoid variants are not (18). In addition, mucoid cells contain hyaluronic acid in their capsules (18). Rough variants have not been found to possess any capsular polysaccharides. Several workers (20, 23) have attempted to determine the dissociation pattern by which these variants arise and the most probably route seems to be: (20): iridescent >mucoid rough rough Various attempts have been made to associate the different variants with different degrees of virulence. Iridescent variants are most often isolated from animals with acute disease while rough and mucoid variants are usually recovered from chronically infected or carrier animals (5, 20). Generally, iridescent variants possess the highest, and rough variants the lowest, virulence for mice (19). Mucoid cultures are usually associated with moderate virulence for mice, although they may vary widely in this respect (l9). Capsular Studies: Nature of Capsular Antigens The capsule of P. multocida is thought to consist of both protein and polysaccharide components (1, 44). Prince and Smith (44) detected two types of capsular

15 antigens, termed a and B. Both were thought to have protein components since both stained with thiazine red. However, trypsin did not affect the precipitation of either, indicating the presence of other substances. The 8 antigen was resistant to boiling, which suggested a polysaccharide component, while a was susceptible to boiling and was precipitable at ph 3.8, indicating that it was mostly protein (44). Briefman and Yaw (3) hydrolysed the capsular polysaccharide and chromatographically identified ribose and galactose. This was unusual in that no other workers had ever reported ribose as a constituent of capsular polysaccharide. Examination of capsular polysaccharides by Knox and Bain (27) revealed nonhomogeneous preparations consisting of 11.2% ketose, 8.2% nitrogen, hexoseamine, fructose, galactose, N-acetylglucosamine and the reducing sugars: glucose, mannose and glucosamine. Ouchterlony tests and ethylene glycol-acetone fractionations indicated the presence of several components in the capsular polysaccharide (27). Because the ratio of fructose: glucosamine varied from one preparation to another, Knox and Bain (27) suggested the possibility of a basic chain containing all of the above-mentioned sugars with varying fructose side chains. The protein-associated capsular substance has been shown to be serologically active, as indicated by its antigenicity and its ability to remove the protective power

16 from antisera (l). The serological activity of capsular polysaccharides has also been demonstrated (3, 27, 43), although immunogenicity of these substances alone has not been observed. These polysaccharides probably act as haptens, which are immunogenic only when associated with protein (27). Knox and Bain (27) failed to observe any protection in mice, rabbits or cattle subcutaneously injected with purified trypsinized capsular polysaccharide when challenged three weeks later. However, these tryp sinized polysaccharides did remove some of the protective power for mice from rabbit and cattle antisera. The a antigen of Prince and Smith (44), which was thought to contain more protein than did the 8 antigen, was also observed to be more antigenic. Capsular Studies: Classification Based on Capsular Antigens Most of the early efforts at a serological class ification of this species employed the use of whole organisms. In 1952, Carter (4) identified three serotypes- A, B and C--by means of precipitin tests with soluble capsular material. Indirect hemagglutination, a more sensitive method, then came into use (6), in which capsular extracts were adsorbed to erythrocytes. By this method, two more types, D (6) and E (12), were identified. However, Carter suggested that his group C be dropped (14) since only very

17 10 few strains fell into this category. His classification, therefore, consisted of four groups (14): A: possessing a wide host range; B: isolated only from cattle and buffalo; D: possessing a wide host range; E: isolated only from African cattle. This classification scheme is still in use at present. It should be noted that most of the mucoid cultures, producing large amounts of hyaluronic acid, fall into Carter's group A. in central Africa. Type E strains have only been isolated Although they cross react to a small degree with type B strains, there are sufficient serological differences to justify their placement in a separate group (12). This was confirmed by Perreau (39). Namioka and Murata (35) confirmed Carter's groups A, B and D by indirect hemagglutination and slide agglutination tests. They observed that the slide agglutination was the less sensitive of the two methods. This was in agreement with Carter (9) who observed that agglutination tests were not suitable for these organisms due to some spontaneous clumping in saline and to an inagglutinability of many capsulated strains, especially if recently isolated. Mouse pretection tests can also be used to group these organisms into capsular groups, although they are not as simple to perform as indirect hemagglutinations. Carter (15)

18 11 observed a linear relationship between the protective capacity of sera, as measured by the logarithm of the number of LD 3 against which 4.25 x 10 2 milliliters 50 of serum protected 50% of the mice, and the reciprocal of the serum titer, as measured by indirect hemagglutina tion. Many studies have been undertaken to determine the capsular types associated with different diseases. Capsular groups A and D are widely distributed with respect to host range and disease (11). Fowl cholera is usually associated with group A organisms (10). Hemorrhagic septicemia of cattle is generally caused by strains of groups B and E. Human infections have been reported, usually as a result of dog or cat bites or entry via the respiratory tract (13). At present, only capsular groups A and D have been recovered from humans (13). However, the role of endotoxin in disease cannot be ignored, since degradation of the capsular in viyg can be expected. The main purpose of such a study of capsular groups is its applicability to vaccination procedures. Type B organisms are employed for bacterin production against hemorrhagic septicemia (8). Bacterins against secondary pneumonia of swine are prepared with groups A and D organisms (8). Heddleston (24) observed that Little and Lyon's (29) capsular types 1 and 3 were capable of

19 12 infecting chickens and that a bivalent vaccine employing both types was necessary for effective protection. Somatic Antigens: Isolation and Characteristics Endotoxin, consisting of lipopolysaccharide with O antigenic side chains, can be isolated by a variety of methods, including phenol-water, aqueous ether, acetic acid and trichloroacetic acid extractions. Endotoxin is part of the cell wall of gram-negative bacteria, probably forming a layer surrounding the mucopeptide of the cell wall. Although no one definite structure for endotoxin has been determined, it is generally thought to consist of a lipid moiety, a backbone of 2-keto 3-deoxyoctanoic acid (KDO), heptose and phosphate (21) and O antigenic side chains composed of repeating sugar components (30). A general scheme of the structure is shown in Figure l. KDO-hep-hep-glu-gal-glu-NAcGlu-[j:} 4 }~4 }.u Lipid -- KDO-hep-hep-glu-gal-glu-NAcGlu-L::k {::}~4;;}v~ moiety w._*f-kdo -hep-hep-glu-gal-glu-nac Glu-E::F { f-{::} L. Y I! L.--_,If V -1- Backbone Core 0 side chains Figure 1. General scheme of endotoxin structure. KDO: 2-keto-3-deoxyoctanoic acid; hep: heptose; glu: D-glucose; gal: D-galactose; NAcGlu: N-acetylglucosamine.

20 13 The lipid moiety is thought to consist of two glucosamine units, substituted with fatty acids. The number of KDO molecules in the backbone is uncertain. O antigenic groups, consisting of specific sugar sequences, are repeated to form 0 side chains. Individual endotoxin molecules are thought to aggregate to form larger structures. Since no chemical assay has been defined by which endotoxin can be identified, biological assays must be employed. Among these are chicken embryo lethality, pyrogenicity and the elicitation of the Schwartzman phenomenon (30). Controversy exists as to the exact location of the toxic portion of the molecule. One of the earliest reports of lipopolysaccharides isolated from P. multocida was by Pirosky (40, 41, 42). These Boivin-like antigens were isolated from both smooth and rough variants. He was able to demonstrate serological differences between various 0 antigens. Type Specific lip0polysaccharides were also isolated by MacLennan and Rondle (31) in Some of the more recent work in this area has indicated that a serological classification of this species on the basis of capsular groups alone is not sufficient. Bain and Knox (2) isolated lipopolysaccharide from Roberts' type I cells by phenol water extraction. They observed that Asian type I strains

21 l4- possessed a different kind of lipopolysaccharide than did the Australian type I cells. Heddleston, Rebers and Ritchie (25) isolated a particulate lipopolysaccharide by successive extractions with cold formalinized saline. They demonstrated that the lipopolysaccharide from an avirulent, noncapsulated avian strain induced active immunity against virulent capsulated organisms in mice, rabbits and chickens. Namioka and Murata's (36) studies of P. multocida indicated the presence of both common and specific 0 antigens. Somatic Antigens: Classification Based on O Antigens Among the first to separate organisms of this species into serotypes on the basis of O antigens were Namioka and Murata (37). Namioka and Bruner (34) were able to separate the Species into ten 0 groups, each designated by an arabic numeral. When 0 and capsular groups were correlated, eleven serotypes resulted: Carter's capsular group Namioka and Bruner's 0 groups A 1, 3, 5, 7, 9 B 6 D l, 2, 3, 4, 10 They acknowledged the difficulty in classification accord ing to O antigens due to the occurrence of multiple cross

22 15 reactions. 1. They also observed the following: Almost all 5:A cultures were isolated from cases of fowl cholera; Most 1:A, 3:A, 1:D and 4:D cultures were recovered from cases of sheep or swine pneumonia; Only groups 5:A and 9:A killed threemonth old chickens within 24 hours; All 0 group 6 cultures were obtained from cattle with hemorrhagic septicemia; Several 0 groups could be subdivided into subgroups. Further studies of the 0 groups of P, multocida by Namioka and Murata (38) indicated the following: 1. A new O group, 11, was observed; 2. Capsular group B strains do not cause hemorrhagic septicemia of cattle unless 0 group 6 is present; ll:b strains do not cause the disease. Fowl cholera is caused by types 5:A, 8:A and 9:A; Types 1:A, 3:A and 7:A are responsible for pneumonia and secondary infections of man and various animals.

23 16 Murata, Horiuchi and Namioka (33) observed that host age affected pathological changes in chickens, even with serotypes that were known to cause acute fowl cholera. Relationship with Other Gram-negative Organisms Several workers have attempted an examination of the relationship between P. multocida and other Pasteurellae by means of taxonomic methods. Talbot and Sneath (49) analysed many characteristics by means of a computer. With the basic assumption that each characteristic carried equal taxonomic significance they observed that Pasteurella pestis and Pasteurella pseudotuberculosis were more closely related to each other than they were to P. multocida. However, a distant relationship did exist. Also using Adansonian taxonomic methods, Smith and Thal (48) divided the genus Pasteurella into two groups: l. Oxidase positive strains, including P. multocida, P. hemolytica and P. pneumotropica; 2. Oxidase negative strains, including 3. pestis and g. pseudotuberculosis. They suggested that the first group be given the genus name Pasteurella and the second group Yersinia. They also

24 17 observed that P. multocida strains were somewhat heterogeneous, although they did show 85% similarity with one another. One other group of workers has tried to study the relationship between P. multocida and other gramnegative organisms. Employing sonic disintegrates and immunodiffusion tests, Prince and Smith (45) studied cross reactions between P. multocida strain 925 and an other gram-negatives: Actinobacillus lignieresi, Brucella suis, Hemophilus canis, Escherischia coli, Neisseria catarrhalis, P. hemolytica, P. pseudotuberculosis and Hemophilus influenza. Cross reactions were observed between P. multocida and all of these strains except B. suis and H. influenza. Since sonic disintegrates were used, many internal antigens, and possibly enzymes, were involved. However, one must consider the possibility that these reactions may have been due to common components of all gram-negatives, such as enzymes and cell wall constituents. The failure to observe cross reactions with two of the other species may have been due to differences in disintegration rates between species.

25 MATERIALS AND METHODS Cultures Twenty-two strains isolated from monkeys were obtained from the National Center for Primate Biology in Davis, California. stock culture medium. Cultures were maintained on Difco All cultures were transferred to fresh stock culture medium at four to five month intervals. Media broth. Blood (ox) agar; serum tryptose agar; nutrient Preparation of Antisera Smooth strains were grown on blood agar and mucoid strains on tryptose agar. Growth was washed off with saline, the bacterial cells collected by centrifuga tion and resuspended in 0.5% formalinized saline. After overnight incubation at 37 C a sterility test was per formed by placing a drop of the suspension into a tube of beef heart infusion semi-solid medium, followed by 24 hour incubation at 37 C. 18

26 19 Immunization schedules consisted of the following: Two rabbits were inoculated with each vaccine. Each rabbit was injected subcutaneously with 0.25 cc of the vaccine with Freund's adjuvant in each of four sites. One month later, 1.25 cc was again inoculated subcutan eously into each of four locations. One month after this, 1.0 cc was injected intravenously (without Freund's adjuvant), followed in four days by 2.0 cc intravenously and four days after this by 3.0 cc intravenously. Four days after the last injection the rabbits were bled out by cardiac puncture. All sera were inactivated at 56 C for 30 minutes and then frozen. Antisera were prepared against five primate strains: MMU 291, MRA 245, ATR 1407, ATR 1403 and MMU Sera to other P. multocida strains were made available by Dr. G. R. Carter. Indirect Hemagglutination Test The procedure used was a modification of the one described by Carter (6). Saline extraction of P, multocida: hour confluent growth from a blood agar or serum tryptose agar plate was washed off with 5.0 cc of physiological saline. This was heated at 56 C for 30 minutes and centrifuged. The supernatant was transferred to another tube for incubation with red blood cells.

27 20 Incubation of red blood cells with extract: Red blood cells were obtained from either chickens or humans and washed three times with 10 cc amounts of physiological saline. All hemagglutination tests described in the Results were performed with chicken erythrocytes unless otherwise stated. 0.1 cc of packed red blood cells were added to each saline extract and incubated at 37 C for 2 hours. The red blood cells were centrifuged out and washed three times with physioloqical saline. Physiological saline was then added to the blood cells to yield a 0.5% suspension. Indirect hemagglutination test: 1:10, 1:20, 1:40 and 1:80 dilutions of sera were made in physiological saline cc of the treated red blood cell suspensions were added to 0.25 cc of the serum dilutions. The tubes were shaken and allowed to stand at room temperature for approximately two hours. A positive reaction was considered to be one in which agglutination was observable in the bottom of the tube, blood cells failed to "run" when the tubes were tilted, and clumping of the hood cells was observable when the tubes were tapped lightly enough to dislodge the cells from the bottom. Control tubes consisted of blood cell suspensions in which the blood cells had been treated with physiological saline instead of with a saline extract.

28 21 Fluorescent Antibody Technique Fractionation of serum: An amount of satur ated ammonium sulfate solution was added to rabbit serum so as to produce a final concentration of 45%. After incubation at 25 C for four hours, collection of the precipitate by centrifugation, and redissolving of the precipitate in distilled water, the same procedure was repeated on the globulin solution. 'The globulin solution was dialysed for 24 hours at 4 C to remove ammonium sulfate. Protein determination: A biuret test was performed on the globulin solution to determine the protein concentration. Conjugation of the globulin with fluoroscein isothiocyanate (FITC): FITC (0.025 mg/mg of protein to be labelled) was dissolved in a volume of ph 9, 0.1 M Na HPO which was half that of the globulin to be 2 4 conjugated. A volume of 0.2 M Na2HP04 equal to one-fourth the volume of the globulin was added dropwise to the globulin, followed by dropwise addition of the FITC solution. so as The ph was adjusted to 9.5 and the volume adjusted to achieve a concentration of 0.05 M NaZHPO4. This mixture was incubated for 2-1/2 hours at 25 C and then centrifuged. The conjugated globulin was dialysed for several days at 5 C against ph 7.5 buffered saline

29 22 until no fluorescence was observed in the dialysate under Wood's light. Borated merthiolate was added to a concentration of l:10,000 and the conjugates were frozen in small quantities. Staining of slides: Chicken red blood cell suspensions treated with bacterial saline extracts were prepared and smears were made on slides, air dried and fixed with light heat. In some cases, heat fixed smears of bacteria themselves were used. The fixed smears were covered with the conjugates and incubated for 15 minutes in a moist chamber at room temperature. The slides were dipped in ph 7.5 buffered saline, washed in ph 7.5 buffered saline for 10 minutes, rinsed in dis tilled water and air dried. Buffered glycerol saline was added prior to addition of a coverslip. The slides were observed with American Optical fluorescence microsc0pe. Capsule Stains Smears from blood agar growth were made in saline with 5% horse serum and fixed in methanol. The slides were covered with crystal violet for one minute, washed and dried, according to the method of Jasmin (26).

30 23 Gel Diffusion Technique Phenol-water extraction of P. multocida: hour confluent growth from a blood agar plate was washed off with 5.0 cc of distilled water. An equal volume of 88% phenol was added and this mixture was heated at 68 C for 20 minutes with frequent mixing. After centrifugation, the aqueous phase was transferred to another tube and to this 10 volumes of cold 0.4 gram % sodium acetate in 95% ethanol was added. This mixture was incubated 1-2 days at 4 C after which the lipopolysaccharide precipitate was centrifuged out and washed twice in cold 0.4 gram % sodium acetate in 95% ethanol. The precipitate was dried, dissolved in 1.0 cc of 1:10,000 merthiolated saline and stored at 4 C. Preparation of Noble agar: Difco Noble agar was dissolved in physiological saline (1 gram/100 cc) by bringing the saline to a boil. The hot agar was immediately filtered through a millipore microfiber glass disc prefilter, Type AP20, and then autoclaved at 121 C for 15 minutes. 1.0 cc of 1% borated merthiolate per 100 cc of agar was added. The agar was distributed in 5.0 cc amounts into 50 x 12 mm disposable petri dishes with tight fitting lids. Six wells (0.5 cm diameter) arranged circularly around one center well (also 0.5 cm diameter) were made in the agar. All wells were one centimeter apart.

31 24 Gel diffusion tests: The lipopolysaccharide obtained from the phenol-water extraction was placed in the center well and the antiserum in the surround ing wells, or vice versa. The plates were allowed to stand at room temperature, and the wells were refilled twice at three day intervals. One or three saline extractions of the bacterial cells at 56 C were, in some cases, performed prior to the phenol-water extraction in order to remove capsular polysaccharide impurities from the final lipopolysaccharide preparations. The procedure was identical to that described for the saline extraction used preparatory to the indirect hemagglutination test, except that the supernatant was discarded and the sedimented cells subjected to phenol-water extraction.

32 RESULTS Table 1 consists of a description of the sources of the cultures which were studied and of the type of colonial variants found. Except for cultures received prior to the start of this project, an attempt was made to determine the major colonial variants present, since only iridescent and mucoid cultures possess enough cap sular material to study by means of serological techniques. Table 2 presents the results of capsular staining. By the Jasmin method of staining, the capsule appears colorless against a light purple background. The bacterial cells stain dark purple. All of the cultures possessed capsules, although the capsules of five strains appeared to be very thin. None of these five strains was typable by the serological methods employed. Preliminary rapid slide agglutination tests were performed, employing erythrocytes treated with saline extracts of the cultures. The results, presented in Table 3, indicate that three of the ten strains examined fell into Carter's capsular group A. 25

33 Source of Specimen Aotus trivirgatus ATR ATR ATR MMU MRA ATR Aotus trivirgatus Macaca mulatta Macaca radiata Aotus tfivirgatus Macaca mulatta Aotus trivirgatus Aotus trivirgatus Aotus trivirgatus Aotus trivirgatus Macaca mulatta Aotus tfivirgatus Aotus trivirgatus Aotus trivirgatus Gum lesion Death; small liver abscesses Death; small liver abscesses Death; small liver abscesses Pneumonia _b _b Small circular lesions Peritonitis; pericarditis Very thin; dehydrated Peritonitis Thin; chronic diarrhea secretions blood blood Nasal secretions Nasal secretions Nasal swab Nasal swab Swab of lesion Nasal swab Nasal swab Swab of lesion Heart blood Heart blood Heart blood Heart blood Heart blood Nasal swab Venous blood Heart blood Heart blood Heart blood Heart blood iridescent 26 TABLE 1 Origin, Associated Disease and Colonial Features of Primate Cultures Colonial a Features Culture Primate Origin Associated Disease Macaca gynomolgus Macaca muiatta Macaca mulatta Respiratory difficulties Death Death MCY MMU MMU ATR Aotus trivirgatus Aotus trivirgatus Macaca mulatta Aotus trivirgatus MMU ATR ATR ATR MMU ATR ATR MMU ATR ATR ATR Aotus trivirgatus _b _b Upper respiratory disease Upper respiratory disease Abscess at tail base Rhinorrhea Rhinorrhea Nasal Heart Heart c Mucoid and iridescent (Blue and mucoid _c Mucoid _c _c Mucoid Mucoid Iridescent and mucoid Mucoid Mucoid Mucoid Iridescent Blue Blue Some Blue Blue _c Blue determined at the time the cultures were received; information not available; not determined.

34 I 27 TABLE 2 Results of Capsule Stains of Primate Cultures Presence of Presence of Culture Capsule Culture Capsule MCY 681 +a ATR MMU ATR MMU ATR ATR MMU ATR ATR ATR ATR ATR MMU MMU ATR MRA ATR ATR ATR 751 +a MMU ATR 906 +a a: Only a very thin capsule was detected. TABLE 3 Results of Preliminary Rapid Slid Agglutination Tests of Saline Extracts Erythrocytes treated Serum with saline extracts of: Type A Type B Type D Type E MCY MMU ATR ATR ATR 843 ATR 849 ATR 1335 ATR 1403 ATR 1405 MMU 5791 u z o z o ND: Not determined Sera used in these tests were: Hull (Type A), 100 (Type B), 37 (Type D), and 33 (Type E).

35 28 Erythrocytes treated with saline extracts of all of the cultures were employed in indirect hemagglutination tests against group specific sera. Table 4 shows the results obtained when several group A sera were employed. Eleven of the cultures gave hemagglutination with group A sera. Serum dilutions were carried out only as far as 1:80, with the exception of Hull group A serum which was diluted only as far as 1:40. Serum dilutions were generally not carried out further than 1:80 because the actual HA titer of the serum was not of as much interest as was the fact that hemagglutination was observed in the lower dilutions. The large number of tests performed also made further dilutions impractical. The other group A sera (MMU 5791, MRA 245, MMU 291, P8, P1059, VA3, 9A) were tested and no hemagglutination was observed. Six of the untypable cultures were passed through seven day old chicken embryos in an attempt to recover organisms with a higher degree of virulence and possibly more capsular material. One tenth of a suspension of cells washed off from a blood agar plate with 5 milliliters of physiological saline was inoculated via the yolk sac. Allantoic fluid was harvested 24 hours later and used to treat human type 0 erythrocytes for use in the indirect hemagglutination tests. Allantoic fluid from embryos

36 29 inoculated with physiological saline served as controls. Five of the six cultures killed the embryos within 24 hours, while the saline inoculated embryos remained alive. However, no hemagglutination could be demonstrated against any of the group A, B, D or E sera tested. TABLE 4 Hemagglutination Tests of Saline Extracts Employing Various Group A Sera Chicken erythrocytes treated with saline Reciprocals of Serum Dilutions Showing Hemagglutination extracts of: Sera: Hull X73 MCY 681 ND MMU 3866 ND MMU ATR 820 ND ATR 848 ND ATR 843 ND ATR 849 ND 40 - MMU MRA ATR MMU 5162 ND ATR 1335 ND 20 - ATR 1403 ND ATR 1405 ND MMU 5791 ND ATR 863 ND ATR 789 ND - MMU 6059 ND ATR 256 ND ATR 442 ND ATR 751 ND ATR 906 ND Untreated ND: Not determined

37 30 Four group-specific fluorescent antibody conjugates were prepared using the following sera: 1403, 100, 2121, 1243, representing groups A, B, D, and B, respectively. One milliliter of each conjugate was adsorbed with the confluent growth from 5 each of the three other groups of organisms. plates The adsorption was carried out for two and one half hours at 37 C. No group-specific reactions could be demonstrated by this method, since all of the organisms employed fluores ced with all four of the conjugates. The use of rhoda mine B as a counterstain did not eliminate the nonspecificity, nor did dilution of the conjugates. However, the conjugates were species-specific, since neither Pasteurella hemolytica nor a Cogynebacterium fluoresced when tested against any of the conjugates. Gel diffusion analysis of the somatic antigens was attempted, employing lipopolysaccharide (LPS) obtained by means of phenol-water extraction as the source of antigenic material. Precipitation due to the reactions between these preparations and the standard antisera against the eleven Namioka O antigenic groups was tested. The results are summarized in Table 5. Three groups of lipopolysaccharide preparations were used. One group of preparations consisted of LPS from phenol-water extraction of the cells. Several of these preparations precipitated

38 31 with many of the antisera. Double and triple lines of precipitation were noticed in gel diffusion plates con taining these preparations. Since there existed a possibility that capsular polysaccharides might be contaminating these preparations, thereby causing extra lines of precipitation, it was decided to perform one or several saline extractions of the cells at 56 C prior to the phenol water extraction. It was assumed that these procedures would reduce the amount of capsular polysaccharide which might be present in the phenolwater extracts and also might expose some of the 0 antigenic sites covered by the capsular material. From Table 5 it can be observed that the number of precipitation lines was reduced by saline pretreatment. In most cases there was little difference in the number of precipitation lines observed after one or three saline pretreatments. However, often different 0 groups were demonstrated in the three LPS preparations of a single culture. The yield of material obtained by each of the three extraction procedures was determined and is presented in Table 6. The yields of most cultures which had been subjected to three saline extractions prior to phenolwater treatment generally ranged from 0.8 to 1.9 milligrams per two plates. The yields per two plates of growth

39 32 TABLE 5 Precipitation Reactions of Somatic Antigens of the Primate Cultures Precipitation with Antisera Precipitation with Antisera LPS to the follow- LPS to the follow- Preparation ing 0 Groups Preparation ing 0 Groups MCY 681: a 1,4 ATR 1335: a 4,5,7,8,9,11 b - b 8 c 1,8,11 c 8,11 MMU 3866: a 5,7,9 ATR 1403: a 4,5,7,8,9,1l b 1 b 8,11 c - c - MMU 291: a 7,11 ATR 1405: a 4,5,7,8,9,1l b - b 8,11 c 1,11 c 7,8,11 ATR 820: a 4,9 MMU 5791: a 1,2,3,4,7,8 b 7,8 b 1,3 c 7 c 1,3,4,ll ATR 848: a 4,9 ATR 863: a 7,8 b 7,8 b 7,8,11 c 7 c 1,7,8 ATR 843: a 7,8 ATR 789: a 4,5,7;8,9,11 b 7,8,11 b 7,8,11 c - c 4,8,11 ATR 849: a 7,8 MMU 6059: a 5,7 b - b l c 8 c - MMU 4531: a 2,3,4,5,7,8,9,11 ATR 256: a 3,4,5,7,8 b b - c c 1,8 MRA 245: a l,3,4,8 ATR 442: a 4,5,7,8,9 b l b 7,8,11 c 11 c 4,8,11 ATR 1407: a 4,7,8,9,11 ATR 751: a 4,5,7,8,9 b 7,8,11 b 7,8,11 0 ~ c - MMU 5162: a 5,7 ATR 906: a 4,5,7,8,9 b 1,2 b 8 c - c - U'DJ LPS from phenol-water extraction; LPS from phenol-water extraction preceded by extraction; LPS from phenol-water extraction preceded by saline extractions. one saline three

40 33 were determined since these were the amounts dissolved in one milliliter of merthiolated saline for use in the gel diffusion tests. No reduction of yields was observed as the number of saline pretreatments was increased. TABLE 6 Lipopolysaccharide Yields of the Primate Cultures LPS Yield (mg/2 plates) No previous One previous Two previous LPS saline saline saline Preparation extraction extraction extractions MCY MMU MMU ATR ATR 848 ND ND 0.9 ATR 843 ND ND ND ATR 849 ND ND 0.2 MMU 4531 ND ND 0.8 MRA 245 ND ND 1.1 ATR 1407 ND ND 1.0 MMU 5162 ND ND 1.0 ATR 1335 ND ND 0.9 ATR 1403 ND ND 0.8 ATR 1405 ND ND 1.2 MMU 5791 ND ND 1.4 ATR 863 ND ND 0.3 ATR 789 ND ND 1.1 MMU 6059 ND ND 1.1 ATR 256 ND ND ND ATR 442 ND ND 0.2 ATR 751 ND ND ND ATR 906 ND ND 1.0 ND: Not determined.

41 34 The lipopolysaccharide preparations in which three saline extractions had preceded phenol~water treatment were employed in chicken embryo lethality tests to determine if one of the biological effects of endotoxin (lipopolysaccharide) could be demonstrated. It is known that salmonella gallinarium endotoxin administered intravenously in the range of to ug is the LD50 dose for eleven day old chicken embryos (32). Eleven day old embryos were inoculated intravenously with 0.02 pg doses contained in 0.05 cc of 0.2% formalinized saline. Control embryos were inoculated with 0.05 cc of 0.2% formalinized saline. Death within 24 hours was considered to be a criterion of endotoxicity. The results are shown in Table 7. Due to the limited number of fertile eggs available at the time these tests were performed, only two embryos were inoculated with each LPS preparation. Six of the eight LPS samples tested killed both embryos within 24 hours. The failure of the two samples to kill both embryos may have been due to an insufficient dosage of lipopolysaccharide. No death was observed in the control embryos. A summary of the findings with respect to the capsular and somatic groups is presented in Table 8. Only the somatic groups observed in preparations which under went three saline extractions prior to phenol-water are

42 35 included in this summary, since it was felt that these samples would contain the least amount of contaminating capsular polysaccharide. TABLE 7 Chicken Embryo Lethality Tests of Lipopolysacharide Preparations of the Primate Cultures LPS Preparation Number of deaths Number of embryos inoculated MMU /2 ATR /2 ATR /2 MMU /2 ATR 863 2/2 ATR 442 2/2 ATR 789 2/2 ATR 906 1/2 0.2% formalinized saline 0/2

43 36 TABLE 8 Summary of Results of Serologic Tests Culture Capsular Group. Somatic Groupa MCY 681 MMU 3866 MMU 291 ATR 820 ATR 848 ATR 843 ATR 849 MMU 4531 MRA 245 ATR 1407 MMU 5162 ATR 1335 ATR 1403 ATR 1405 MMU 5791 ATR 863 ATR 789 MMU 6059 ATR 256 ATR 442 ATR 751 ATR 906 >35>Svki>fiibfiab I Ital lml\l\l) ' " H I bkdl bkdhhdl m I,8,ll V I-" H Q H H ~ mcn m~4uam f l - Idaabra H H ~ H H H a: Corresponding to Namioka's somatic groups.

44 DISCUSSION Of the eleven cultures whose capsular groups were determined, all were observed to be group A strains. Since strains of groups B and E have only been isolated from cattle, it was suspected that the primate strains would possess either A or D capsular antigens. The remaining eleven strains could not be typed by the serological methods employed. Several possibilities exist which may explain this failure to detect capsular antigens. Of the eleven group A cultures, most consisted of mucoid or iridescent variants at the time these studies were initiated, indicating an amply supply of capsular material. In contrast, many of the remaining cultures were composed of rough variants, possessing relatively small amounts of capsular substances. Therefore, the absence of adequate amounts of capsular antigens would prevent their detection. Most of the eleven cul tures classified as group A lost their ability to cause indirect hemagglutination after maintainance and transfer in stock culture medium for one year, indicating that dissociation to the rough variants had probably occurred. The presence of other unknown capsular groups within the species may also have been responsible for the inability 37

45 38 to type these cultures on the basis of their capsular antigens, although this possibility seems to be less likely. Many attempts were made to study the untypable cultures. Passage of these cultures through chicken embryos was performed in an effort to increase the numbers of virulent, capsulated organisms. Both chicken and group 0 human erythrocytes were employed in indirect hemagglutination tests. However, none of these efforts were successful. Treatment of the saline extracts of mucoid cultures with hyaluronidase was also attempted in order to remove the nonantigenic hyaluronic acid from the material and possibly expose more of the capsular antigens, but these efforts were also unsuccessful. Tanning of the erythrocytes was employed in order to determine whether indirect hemagglutination could be demonstrated by allowing some of the protein of the capsular material to adsorb to the erythrocytes. However, this procedure was also ineffective in producing hemagglutina tion with the untypable cultures. It was hoped that the fluorescent antibody technique would present a rapid means of identification of the different capsular groups. The inability to obtain type specific fluorescein conjugates within this species, even after adsorption, seems to indicate the presence of

46 39 many common surface antigens. NonSpecific staining due to technical reasons does not seem likely since organ isms of other species, including the closely related Pasteurella hemolytica, did not fluoresce after treatment with the conjugates. The complexity of the antigenic makeup of Pasteurella multocida was demonstrated by analysis of the somatic antigens. Antisera representing Namioka's eleven somatic types were used. Each of these antisera was prepared against intact formalinized organisms, since purified lipopolysaccharide is poorly immunogenic. It was assumed that degradation of some of the surface components, including the capsule, occurred within the host, thereby exposing some of the somatic antigens. Namioka (36) regarded his somatic classifications as groupings based on antigenic complexes composed of several factors. In this case, each somatic designation may represent the presence of several 0 factors. The antisera used in this study were not adsorbed with organisms of the ten heterologous types and therefore the reaction of a single lipopolysaccharide preparation with several antisera, as is observed in Table 5, may be due to cross reactions. The chemical composition of the 0 factors corresponding to each somatic designation has not as yet been examined and thus the antigenic determinants involved are not known.

47 40 Table 8 indicates that the primate strains reacted mainly with antisera to four of the somatic groups: 1, 7, 8 and 11. In some cases the lipopolysaccharide from a single strain reacted with several of these antisera, possibly due to cross-relatedness of the antigens of these groups. Adsorption of each of the four antisera with organisms of the three heterologous types might clarify these results somewhat. Lipopolysaccharide from several strains gave no precipitation with any of the antisera. Mutations resulting in loss of O antigens might be responsible. It should also be noted that the number of saline extractions performed prior to phenol water treatment affected the kinds of 0 groups observed. For cultures receiving no saline pretreatments, the presence of capsular antigens may have been responsible for some of the large number of precipitation lines. Each saline pretreatment prior to the phenol-water extraction probably removed more of the surface components from the cell. Further study is necessary to understand the nature of the capsular and somatic antigens of P. multocida and their role in infection. The numbers of these antigens remains unclear. Prince and Smith (44) have found two types of capsular material in all P. multocida organisms they have studied. Working with a single strain of organ isms they demonstrated the presence of sixteen other

48 41 soluble antigens. Since these were detected in sonic disintegrates of cells, it is not known how many of these are 0 antigens. The number and kinds of common antigens also remain unknown. In summary, eleven of twenty-two monkey strains were found to possess group A capsular antigens by means of indirect hemagglutination tests. The four main somatic antigenic types observed by precipitation reactions in gel agar were 1, 7, 8 and 11. The fluorescent antibody technique was ineffective in this serological typing since type specific fluorescein conjugates of antisera could not be prepared.

49 BIBLIOGRAPHY

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55 I I I I I I I I I[I I I I I I I I I IEI ITS