“1“, . \ ..a_..' ‘ . .,,. x' ‘. . _ - a...‘ ‘v' I.'.\ ;.'.-‘. ‘9‘.“ 8;},‘335'1‘4 .3. K -'- !‘(. \. I. . _' - . '. - -‘- '.‘ ' ' . - ~ . ' . I. ' ’I’l 1; ,V.:."“ ‘ . '..,..:.9"",'I" ‘I'I‘ ’ ~"O.j.'_‘ ' ‘ I I , ‘. ‘ '- . , u . v I, . n - t '_ ‘ “f"! ' , ' O n 5",.4 ‘5“{' ,-‘~"‘ ‘l‘ .‘3 . . - ‘ . ‘- " '. .' '~_ - _ -. ' , . ’ seaowesc STUDIES on some smms or PASTEURELLA MULTOCIDA Thesis for the Degree of M. S. MIOHI‘GAN STATE UNIVERSITY 'SUB—RONTO .PRODJDHARJONO 1972 ' LIBRARY IIIIIII IIIIIIIII IIIIII IIIIIII I/ meme. 3129 01009 l1"043 uniVCIfity DEC! 53902;: ABSTRACT SEROLOGIC STUDIES ON BOVINE STRAINS OF PASTEUHELLA MULTUCIDA By Subronto Prodjoharjono An attempt was made to identify_the serotypes of 40 bovine strains of Pasteurella multocida isolated from several areas of midwestern United States. The indirect hemagglutination (IHA) test was employed to identify the capsular antigens of the strains, while the rapid . agglutination (RA) test was used for the somatic antigens of the strains. Thirty-six strains (90%) were found to belong to type A and four (10%) to type D (Carter). Thirty strains possessed somatic anti- gens group 1 (Namioka and Murata) with different subgroups, viz., a, b, and c. The somatic antigens of 10 strains were not satisfactorily identified. To avoid cross reactions attributable to "normal antibody" in sero- logic studies involving P. multocidh, examination prior to the inocula- tion of antigens is recommended. The tests for antibodies to P. multocidfi in the bovine sera did not provide evidence to indicate that a single dose of the PasteureZZa bacterin produced a significant humoral immune response. Application of the microtiter system for performance of the IRA test was shown to be practicable. SEROLOGIC STUDIES ON BOVINE STRAINS 0F PASTEURELLAIMULTOCIDA By Subronto Prodjoharjono A THESIS Submitted to Michigan State University‘ in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Large Animal Surgery and Medicine 1972 Dedicated to my family Ien Subronto, Hari, Adi, and Janri ii ACKNOWLEDGMENTS I wish to extend my sincere appreciation to all those who willingly contributed their time and interest in making this study possible. Special notes of thanks are directed to Professor Dr. G. H. Conner, my academic adviser, for his patient guidance, continuous assistance, counseling, and especially encouragement; Professor Dr. G. R. Carter, for his unceasing guidance, constructive criticism, disciplined thinking, and encouragement; Professor Dr. F. H. Oberst, Professor Dr. K. K. Keahey, Professor Dr. J. T. Huber, and Professor Dr. A. L. Trapp, for their willingness to serve in the guidance and examining committee. Their careful examination of this thesis is greatly appreciated. I am also indebted to Dr. D. P. Olson for his kindness to supply the blood samples and his cooperation in discussing the results of the vaccination. The staff members of the Clinical Microbiology Laboratory are impossible to forget for their assistance and jovial encouragement. My family, far away in Indonesia, gave their full moral support and patience. To them this study is dedicated. Lastly, to the MUCIA-AID-INDONESIAN HIGHER AGRICULTURAL EDUCATION PROJECT, which has financed my study in the U.S., and all others who contributed in any manner, my sincere thanks. iii INTRODUCTION. TABLE OF CONTENTS REVIEW OF LITERATURE. o o o o o o '0 o o o '0 o o o ‘o o o o to Nomenclature of P. multocida . . . .-. . . . . . . . Animal Species Affected by P. multocida. . . . . . . Cultural Variation of P. multocidh . . . . .'. . . .- Classification Based on Biochemical Reactions. . . . Serologic Classification of P, multocidb . . . . . . MATERIALS AND Typing Early Investigations. .A. . . . . . . . . . . Recent Investigations . . . . . .'. . . . . . K or Capsular Antigen.of P. multocidb . . . . O or Somatic Antigen of P. multocida. . .-. . Serologic Studies of Bovine Pasteurellosis. . MTHODS O O O O O O O O O O I I O O O O I O O O O ‘ of Bovine Strains of P. multocidb . . . . . . Strains of P. multocidh. . . . .-. . . . . . Media . . . . . . . . . .‘. ._. .'. . . . . . Preparation of Antisera . . . . . . .‘. . . . Performance of Indirect Hemagglutination Test Rapid Agglutination Test. . . . . . . . . . . Acriflavine Test. . . . . . . . .‘. . . .-. . Detection of P. multocidh Antibodies in Bovine Sera. RESULTS . . Preparation of the Serum. . . . . . . . . . Indirect Hemagglutination (IHA) Test. . . . . . . Indirect Hemagglutination Test Employing a Micro- titer TeChnique. O O O O O O O O O O O O 0 Rapid Agglutination Test. . . . . .'. . . . . Part 1 o o o o o o 'o o a o I. o o o o o o o o o o o o Antibody Titers of Rabbit Antisera. . .-. . . Antisera Employed for the IRA Test . .‘ Antisera Employed for RA Test. . . . . iv 5 Page .11 -12 13 - 14 18 - 18 18~ 18 18 21 22 22 23 23 24 24 24 25 25 25 ~ 25 26 Page Capsular Types Determined by Indirect Hemagglutination Test . . . . . . . . . . . 26 Results of Examination of Strains with the Rapid Agglutination Test. . . . . . . .- 26 Part 2 o o o o o o o o o o o ‘0 o o o o o I. o o o I. o o Occurrence of Antipasteurella Antibodies in Bovine Sera. . ; . . . . . . . . . . .'. . . . . ., 29 Antibodies Measured by the IHA Test: Prevaccination.~. . . . . . . . . . . . . .' 29 Antibodies Measured by the IHA Test: Postvaccination . . . . . . . .-. . . . . . 29 Antibodies Measured by the Rapid Agglutina- tion Test: Prevaccination. . . . . . . . . 36 Antibodies Measured by the Rapid Agglutina- tion Test: Postvaccination . . . . . .-. . 36 Antibodies to P. hemolytica Type I Measured by the IRA and the Agglutination Tests. . . 36 DISCUSSION. . . . . . . . . . . .-. . . .'. . . . . . . . . . . . . 45 Part 1 . . . . . . . .-. . . . . . . .-. . . .'. . . . . . . 45 Part 2 . . . . . .'. . . . . . . . . ._. .'. . . . . . . . . 48 SUMMARY . . . . . . . . . . . . . .-. . . .-. . . .'. . . .-. . . . 50 BIBLIOGRAPHY. . . . . . . . . . . ._. .~. . . . . . . . . . . .'. . 52 “PENDIXO 0 O O O O O 0 .0 O O O '0 O O O .0 O O O '0 O O O .0 O O O V. O 58 Table 10 11 12 13 14 15 LIST OF TABLES Proposed and older designations for colonial variants 0f Pasteurezza muZtOO’ida. o o o o o o o o o o '0 o o o 0 Some characteristics of Pasteurella multocida and their relationship to colonial variants . . . . . .-. . . . . Early serologic studies on Pasteurella multocida. . . . Serotypes of bovine strains of Pasteurel7a multocida isolated from several countries . .'. . . . . . . . . Source of the bovine strains of Pasteurella multocida . Titers of antisera employed in the IHA test . . . . . .- Typing sera employed-for the rapid agglutination test . Titers of antisera and homologous antigens in the rapid agglutination test. . . . . . . . . . . . . . . . . . Capsular types of bovine strains of P. multocida determined by IHA test 0 C O O O C C O O O O 0 O C O O 0 Results of rapid agglutination test with selected 0 group ant 18era O O O O O O O O O O O O O O O O O I O O O Serologic identification of bovine strains of Pasteurella multocida . . . . . . . . . . . . . . . . . Pasteurella antibody titers employing IHA test in cattle prior to vaCCination 'o o o o o o o o o o o o 'o o Pasteurella antibody titers employing IHA test using microtiter technique (strain B-l476-71 as antigen) in cattle prior to vaccination . . . . . . . .‘. . . . . . Pasteurella antibody titers (IHA test) in sera collected from control animals 2 months after the initial test using strain B-1476- 71 as antigen . . . . . . . . . . . Pasteurella antibody titers (IHA test) in sera collected from vaccinated animals 2 months after the initial test using strain B-l476-71 as antigen . . . . . . . . . . . . vi Page 11_ 16 19 25 26 27 - 28 3O 31 32 34 37 38- Table Page 16 Pasteurella antibody titers (IHA test:' microtiter technique) in sera collected from control animals 2 months after the initial test using strain B-l476- 71 as antigen. . . . . . . . . . . . . . . . . . . . . . . . 39 17 PasteureZZa antibody (IHA test: microtiter tech- nique) in sera collected from vaccinated animals 2 months after the initial test using strain B- 1476-71 as antigen. . . . . . . . . . . . . . . . . . . . . . . . 40 18 PasteureZZa antibody titers employing rapid agglutina- tion test in cattle prior to vaccination, using strain B-702 (0 group la,b) as antigen . . . . . . . . . . . . . 41 19 PasteureZZa antibody titers (RA test) in sera collected from control animals 2 months after the initial test using strain B-702 as antigen . . . . . . . . . . . . . . 43 20 PasteureZZa antibody titers (RA test) in sera collected from vaccinated animals 2 months after the initial test using strain B-702 as antigen . . . . . . . . . . . . . . 44 A-l Pasteurella antibody titers (RA test) in unimmunized rabbits 0 o o o o o o o 0'. o o o o o o o o o o o o o o 0' 58 A—2 Pasteurella antibody titers (RA test) in unabsorbed and absorbed antisera o o o o o a". o u o o o o o o o o o o o 58 vii INTRODUCTION The organisms causing hemorrhagic septicemia in cattle, swine, buffaloes (Barbone), fowl cholera in chickens, pneumonia, and diseases in other animals including swine, cats, rabbits, sheep, and horses have r‘fi received considerable attention since the turn of the century. It has been known for many years that the maladies caused by these organisms I occur widely in many parts of.the world. Earlier observations.of Bolinger, Kitt, Hueppe, Lignieres, Manninger in EurOpe, and Moore, L Smith, Mohler, and EiChhorn in the United States, indicated that many species of animals were susceptible to the organisms which later were given the name Pasteurella multocidh. Considerable research dealing with the pasteurelloses has been-carried out. Since Lignieres (1900) prOposed his well known zoological classification of Pasteurella, other classifications have been presented in order to clarify the multicom— plexity of these organisms. Division based upon cultural variation, biochemic, and serologic characteristics has been proposed by several research workers. The schema of Roberts (1947) was based on the.mouse protection tests, and that of Carter (1955) on the specificity of the capsular antigen., Attempts to produce vaccines and bacterins to prevent pasteurellosis have been made by Pasteur, Kitt, Lignieres, and others. Mohler and Eichhorn.(1912), in their effort to combat an outbreak of hemorrhagic septicemia in bison in Yellowstone National Park in 1911, employed Lignieres' immunizing method. Two strains of P. multocidh obtained from 1 2 the outbreak and from cattle near the outbreak were used to prepare the vaccine. To evaluate the efficacy of vaccination, they employed a complement fixation test and claimed that this method could be employed to diagnose the infection as well. They were successful in preventing future outbreaks, and it was concluded that they had eliminated the disease.- With one exception (Kradel et al., 1969), no cases of hemor- rhagic septicemia have been reported in the United States since. There is some question as to the origin of the strain that resulted in the one small episode in 1968. It is interesting that the bison strains which were still extant resemble closely the Asian hemorrhagic septi-- cemia strains. Bain (1955) in evaluating vaccination in cattle and water buffaloes in Thailand concluded that the mouse protection test was superior to agar agglutination, agglutination, and complement fixation tests.' Perreau et al. (1964), using the passive hemagglutination procedure and the mouse.protection test, examined 411 samples of sera collected from the Republic of Chad, Africa. He found that 82 samples possessed a con- siderable amount of specific antibody to P. multocida type E. In this study an attempt was made to determine if the vaccination of cattle with-Pasteurella bacterin resulted in the production of spe- cific antipasteurella antibody. The indirect hemagglutination (IHA) test'and the rapid agglutination (RA) test were used for this purpose and also to identify the serotype of the strains of P. multocidh collected from several areas of midwestern United States. REVIEW OF LITERATURE Nomenclature of P. multocida In 1877 Rivolta reported a member of the PasteureZZa group as the etiologic agent in septicemia epizootica. Later veterinary bacteriolo- gists Bollinger, Kitt, and Hueppe uncovered the multicomplexity of these organisms as disease agents for several species of animals. Kitt in 1885 used the name Bacterium bipolare-multocidum instead of the name. Bacterium septicemia-hemorrhagica which was employed by Hueppe in 1886. The latter was the first to note the close relationship of various organisms.of the group which included causal agents of fowl cholera, cattle, rabbit, and swine septicemia. Based upon the history of the isolation of members of the Pasteurella group from several animals, Lignieres (1900) proposed the well-known zoological classification of the PasteureZZa genus. Strains were named according to the host animals, e.g., PasteureZZa boviseptica. PasteureZZa aviseptica, PasteureZZa suiseptica, etc. Six groups of pasteurellosis have been proposed, viz., Pasteurellose aviaire, Pasteurellose porcine. Pasteurellose ovine, Pasteurellose bovine, Pasteurellose equine, and Pastuerellose canine. Even though experimental evidence showed some inadequacy in this method of classification, it was accepted and employed for many years.‘ Rosenbusch and Merchant (1939), in their review, pointed out that Baumgarten (1911), Mohler and Eichhorn (1913), Hutyra (1928) and Manninger (1934) had raised objections to Lignieres' classification. Based on cultural, biochemic, and serologic characteristics the 3 4 Pasteurellae have been divided into two groups, viz., typical and atypical. The-atypical group has been studied in detail by Newsom and Cross (1932) and, on the bases of pathogenicity, ability to produce hemolysis, absence of indol formation, and fermentation of lactose, maltose, inositol, dextrin, and raffinose, was found to be identical to Jones' Group I. They also proposed the name Pasteurella hemolytica for the atypical group. Rosenbusch and Merchant (1939), using the criteria of cultural, biochemic, serologic, and pathogenic characteristics,.proposed.reclassi— fication of the PasteureZZa groups causing diseases in animals into two major.species, viz., P. multocidb and.P. hemolytica. The first species was to be divided into two distinct SUbgroups and a third less distinct one on the bases of fermentation of xylose, arabinose, and dulcitol, and on agglutination reactions. Animal Species Affected by P. multocidb It has been known for many years that P. multocida can be isolated from many species of animals. Smith (1891) reported that they could be found in the throats and air passages of what appeared to be normal - swine. Moore (1893) noted that pasteurellas were pathogenic for rabbits and.that the carrier incidence of apparently normal hogs was 48%, and in cattle 80%.' Jorgenson (1925) reported isolation of P. boviseptica from 37 of 250 cattle he examined. Smith (1955) recovered P. multocida from the tonsils (54%) and nasal passages (10%) of 111 dogs. In his review to 1955, he noted that carrier rates in normal cattle and buffaloes varied from 3.5 to 72 and in cats it ranged as high as 90%. Carter (1959) also obtained a large percentage of P. multocida from the gum lines of cats he examined. 5 Bakulina (1961) isolated Pk multocidafrom.gerbils, Golebiowski- (1961) from a coypu, a dove, a peacock, a cormorant, a pelican, and a vulture, Mlinac (1961) from nutria, Post (1962) from Rocky MDuntain- bighorn sheep, Ponomareva and Rodkevich (1964) from moles, and Young (1965) from kangaroo. In human beings,Ph multocida has been recovered from 2 of 71 veterinary students (Smith, 1959). Heddleston (1966) reported that he possessed high agglutination titers for several strains after working with this species for many years. He also recovered P. multocida from his throat. Olsen and Needham (1952) found that the majority of cases in man occurred in pe0p1e who had ample opportunity for contact with infected animals. Cultural Variation of P. multocidb The earliest report dealing with cultural variation was made by Manninger (1919), who described an avirulent, uncapsulated, highly immunogenic variant of avian origin.‘ De Kruif (1921, 1922) described the mutants of P. multocida as type D (diffuse) for virulent strains and type G (granular) for avirulent and rough strains. The type D variants produced a diffuse growth in broth, while the type G produced a granular deposit. Webster and Burn (1926) prOposed the designation I, or Intermediate, for the unstable type and M, or Mucoid, for the more stable type. Anderson et a1. (1929) used the designation S (smooth) and R (rough) to replace D and G of De Kruif. Other culture variants were also noted by Webster and Hughes (1929) and by Hughes (1930). They correlated their fluorescence (iridescence), pathogenicity and agglutinability, and found that the fluorescent form was highly virulent and of poor agglutinability 6 while the non-fluorescent (blue) form was found to have lower virulence and high agglutinability. The Intermediate form varied in character- istics between the two aforementioned forms. Smith (1958) observed only small differences in the morphology of PasteureZZa cultures from cattle, swine, dogs, and cats. The various designations used in earlier studies were reviewed and summarized by Carter (1957a) (see Table 1). Table 1. Proposed and older designations for colonial variants of Pasteurella multocida* PrOposed terms Colonial Antigenic Corresponding terms applied earlier Mucoid M Mucoid (Wébster and Burn, 1926) Smooth S Fluorescent (Hughes, 1930) Smooth 8 Intermediate (Hughes, 1930) Smooth SR Granular (De Kruif, 1921) Blue (Hughes, 1930) Rough R Rough (Anderson et aZ., 1929) * Carter, 1957a. Following the recommendation of Braun (1965), Carter (1957a) used the acriflavine test and recommended the terms mucoid, smooth, and rough to designate the principal variants of P. multocidh. The most frequently. seen were mucoid, smooth (iridescent), and smooth .(noniridescent); their relation to hemagglutinability and acriflavine-reaction are shown in Table 2 (Carter, 1957a). With the exception of type D strains, it_was observed that variants that flocculated strongly in the acriflavine test 7 did not possess sufficient specific capsular substance and were designated as SR. Table 2. Some characteristics of Pasteurella multocida and their rela- tionship to colonial variants* Colonial Antigenic Typable by indirect variant designation Capsule Acriflavine hemagglutination Mucoid M ++** Slimy + Smooth S + Cells remain + suspended R . Smooth S - Flocculation - Rough R - Flocculation - * Carter, 1957a. * Estimate of degree of capsulation. Besides studies based on cultural characteristics of P. multocida and their pathogenicity and agglutinability, attempts have been made to classify these bacteria based on the biochemical activities in culture media. Classification Based on Biochemical Reactions Smith (1891) noted that all of the Pasteurella strains he investi- gated were able to ferment dextrose and saccharose but failed to produce acid in milk or lactose. He also recognized that a close resemblance existed between Bacillus bovisepticus and Bacillus suisepticus. SchirOp (1908) recognized a slight difference between the strains of Bacillus vitulisepticus recovered from calves. Two strains fermented 8 dextrose and saccharose but not mannitol, while the other strain fermented only dextrose. Magnusson (1914), who worked with bacteria recovered from a septicemia hemorrhagica outbreak among reindeer in Lappland, Sweden, noted that these bacteria had biochemical reactions similar to Bacillus bovisepticus and Bacillus avisepticus. They pro— duced acid in lactose, saccharose, and mannitol but not in maltose. WOrking with 16 strains of Pasteurella isolated from cattle with pneumonia, Jones (1921) divided Pasteurella into 3 well defined groups onrthe basis of biochemical aetivity, viz., Groups I, II, and III. Later Jorgenson (1925), working with 37 strains isolated from 250 cattle, added a fourth group to the Jones' classification. The latest group had the ability to ferment dulcitol, and was extremely virulent in nature. Other classifications based on biochemical differences were pre- sented by Khalifa (1934).- His pasteurellas were divided into 3 types, viz., types A, B, and C. Type A had the ability to ferment mannitol and arabinose, type B fermented xylose, and type C split mannitol and xylose.v Inconsistency in the biochemical reactions of P. multocida apparently was the main reason that workers considered other classifications. Among others, Rosenbusch and Merchant, Ochi, and Roberts used biochemical dif- ferences to support their serologic classifications. Serologic Classification of P. multocida Early Investigations The zoological classification of Lignieres was not satisfactory due to the antigenic diversity among the pasteurellas. Similar or anti- genetically identical strains were found in several different animal hosts.' 9 Tanaka (1926), employing agglutination and complement fixation tests, was not successful in his attempt to group septicemia hemorrhagica bacteria. Both methods had failed to demonstrate specific immunological characteristics of the strains examined. Cornelius (1929) performed agglutinin-absorption tests and divided his strains into 4 groups, viz., Groups I, II, III, and IV. He found no serologic relationship with host specificity. Nine of 26 strains he examined defied classification. Later precipitation-absorption tests were conducted in the same laboratory by Yusef (1935). Of 21 strains used by Cornelius, 14 had been classified as Group III (Cornelius). It was assumed that his results were comparable to those obtained by the agglutinin-absorption technique. By means of cross agglutination tests employing the variants of the strain as antigen, Ochi (1933) succeeded in differentiating 4 types of P. multocida designated as types A, B, C, and D. Type A strains were obtained from fowl and were pathogenic for laboratory animals and birds, while calves and pigs were resistant. Type B recovered from cattle, swine, and buffaloes was pathogenic for laboratory animals, calves, and pigs, but not for birds. Type C strains recovered from swine, sheep, and.mice possessed pathogenicity comparable to type B. Type D strains were from sheep, and were avirulent for mice. The existence of these types was confirmed by Zaizen (1934) employing complement fixation tests on the strains isolated by Ochi. Later Ochi at al. (1942) found that his type D differed serologically and biochemically from the other types and corresponded to P. hemolytica. Pirosky (l938a,b), working with 4 strains, found that they consisted of 2 distinct serologic varieties. He employed cross precipitation tests using Boivin type antigens and sera prepared by inoculating 10 rabbits with rough and smooth variants derived from the-same strain of P. aviseptica. 0n the basis of fermentation of xylose, arabinose, and dulcitol,' Rosenbusch and Merchant differentiated P. multocida into 3 subgroups (vide supra). 'They showed no consistent serologic relationship with host specificity.* Rapid agglutination tests with Specific antisera and passive immuni- zation of mice were used by Little and Lyon (1943) to confirm the existence of the 3 serologically distinct-types of P. multocida, viz., types 1, 2 and 3.‘ The specificity of each type was demonstrated by mouse protective tests against at least 10,000 lethal doses of the virulent cultures. The monovalent antisera types 1, 2 and 3 which were obtained by serial injections of rabbits protected mice from death due to the organisms of homologous type. Another classification based on immunological differences has been proposed by Roberts (1947). Supported by a number of cultural and bio- chemical differences in employing cross protection in mice, 37 strains of.Pasteurellae have been classified into 4 types, viz., types I, II, III, and IV. It was claimed that his classification had a loose rela- tion to host species. He also suspected that there might be'7 or 8 types in his collection. A type V has been added to the Roberts' classification by Hudson (1954) after observations indicated that some porcine strains differed from the types I-IV in their growth in blood media containing xylose. The designations used in early serologic studies were compared and summarized by Carter (1967) (see Table 3). 11 Table 3. Early serologic studies on Pasteurella multocida* Investigators Procedure Results Cornelius (1929) Agglutinin absorption Groups I, II, III and IV Ochi (1933) Agglutination Types A, B, C, and D Yusef (1935) Precipitation Groups I, II, III and IV Rosenbusch and Agglutination, Groups I, II and III Merchant (1939) fermentation Little and Lyon Slide agglutination Types 1, 2, and 3 (1943) Roberts (1947) Serum protection tests Types I, II, III and IV in mice * Carter, 1967. Recent Investigations A precipitin test employing saline extracted capsular subatances gave promising results in the hands of Carter (1952). He identified 3 groups or types of P. multocida, viz., types A, B, and C; however, since the tests required considerable amounts of serum and antigen and were subject to some cross reactions, another method was developed. This was the indirect hemagglutination (IHA) test, in which capsular antigen was adsorbed to red blood cells (Carter, 1955). With this method P. multocida strains were divided into 4 groups or types, viz., types A, B, C, and D.‘ It is now known that Roberts' type I strains are identical to Carter's type B; types II, III, and IV are probably type A and type V was identi- fied as type D (Carter, 1972a). Strains recovered recently from bovine septicemia hemorrhagica cases in Central Africa have been identified as a new serotype, viz., type E (Carter, 1961).- Contemporaneously, Perreau (1961), employing IHA 12 tests, agar—precipitin and mouse protection tests, confirmed that strains obtained from the above area differed serologically from Carter's type B. Because of difficulties in identification, type C category was drapped from the classification (Carter, 1963a). Namioka and Murata (1961a) employed slide agglutination tests as a simplified-method to type P. multocida. They claimed that if the cultures used were of the fluorescent type, the results were parallel to those of the IHA tests. They also pointed out that the IHA test was the most reliable procedure for obtaining accurate results in typing the capsule of P. multocida. Carter (1963b) found that the slide agglutination test was not a reliable substitute for the IHA test.‘ Slide agglutination tests for the identification of capsule types were conducted by N¢rrung (1963).- He succeeded in typing 28 of 45 strains; 17 strains remained unidentified because of inagglutinability or cross reactions. K or Capsular Antigen of P. multocida Priestley (1936) claimed that Manninger in 1919 was the first to demonstrate a relationship between the capsule and virulence of P. multocida. Smith (1927) noticed that freshly isolated bacteria were heavily encapsulated. The capsule was found to be diminished after varying periods of laboratory cultivation. He also obServed some correlation between capsulation and virulence of the bacteria, as did previous workers. The "Kapselsubstanz" extracted-chemically from Bacillus avisepticus (Hoffenreich, 1928) had the ability to react specifically with the immune sera, although it did not produce immunity in rabbits. 13 Carter (1952), in his effort to classify P. multocidb on the basis. of capsular antigen specificity, divided the strains into 3 types, viz.. A, B, and C‘(vidc supra). Only nonmucoid strains were typable with this early procedure.v The mucoidness of the type A strains later was found to consist in large part of hyaluronic acid (Carter, 1958;~ Mannheim, 1961). Treatment with the enzyme hyaluronidase has given effective results in preparing antigen of types A and D strains for the IHA test (Carter, 1972b). To date, types A, B, D, and E are the types found among the strains of P. multocida. Because of difficulties encoun- tered in detecting type C strains this category was eventually drapped (Carter, 1963a). The presence of additional capsular types remains a real possibility (Carter, 1967). Carter (1967), in his comprehensive review article, summarized the major antigenic components into 4 complexes, viz., polysaccharides, hyaluronic acid, lip0polysaccharides, and protein. 0 or Somatic Antigen of P. multocidg It has been known for many years that cross reactions occur in serologic studies. Precipitation and agglutination tests might lead to misinterpretation if they were not conducted with the "pure" monovalent antigen and antiserum. Namioka and Murata (1961b), in their elaborate studies, treated their strains with 1N HCl to render them agglutinable. They produced 0 antisera by intravenously inoculating rabbits 15 times at 5-day intervals. They employed an agglutination absorption technique. In their summary, they stated that 0 antigen did not change readily into rough antigen.when cultures were subcultured for a year or more.' By 14 absorption tests, the O antigens of P. multocida were divided into 2 antigens, common and specific. In other studies, Namioka and Murata (1961c), employing absorption tests on 24 strains, concluded that there were 6 0 groups and, by com- bining them with the capsular antigens, 6 serotypes were designated. They also proposed that serotypes be identified by first listing the number standing for specific 0 antigen followed by a capital letter representing the specific K antigen, e.g., 6:B. Namioka and Bruner (1963), in their investigations of 156 strains of P. multocidh, found 10 0 groups. By correlating these 0 groups with Carter's capsule types 12 serotypes were delineated. To date, 11 dif- ferent 0 groups have been identified (Namioka and Murata, 1964). Serologic Studies of Bovine Pasteurellosis. The occurrence of pasteurellosis in cattle has been known for many years. Pasteurella multocida has been incriminated as the sole.etiologic agent of septicemia hemorrhagica or hemorrhagic septicemia and as a part of the etiology of the shipping fever complex. Ample research dealing with the role of P. multocida in these maladies has been carried out in many parts of the world. Earlier serologic classifications based upon precipitation, agglutina- tion, and complement fixation tests provided very little useful informa- tion on bovine strains. The serum protection tests in mice carried out by Roberts (1947) proved a useful method for identifying serotypes of P. multocida. The bovine strains were found in all 4 types or categories of Roberts. Bain (1954a), employing Roberts' method, found that 23 of 24 South East Asian 15 Pasteurella strains recovered from septicemia hemorrhagica in cattle or water buffaloes belonged to Roberts' type I. Roberts pointed out that monovalent antigen or antiserum was of little value in bovine pasteurellosis of the more common pneumonic type. Despite the difficulty and expense of combining antigens he recommended a bivalent bacterin prepared from his types I and II to prevent pasteurel- losis outbreaks in cattle and pigs. Natural immunity against septicemia hemorrhagica in buffaloes has been observed by Bain (l954b,c) in Thailand by means of agglutination procedures. Application of those tests in experimental buffaloes has also been carried out to evaluate the vaccination. In other studies, in his attempt to evaluate the efficacy of vaccination in cattle using "Insein" strain, he found that the mouse protection tests gave the best indication of immunity in vaccinated animals. Agar agglutination tests, agglutination tests, and complement fixation tests have been found to be less satisfactory (Bain, 1955a,b). Carter (1955) employed an indirect hemagglutination test to identify different capsular types.of P. multocida. All 4 types, viz.,-types A, B, D, and E, were found occurring in bovine pasteurellosis. Type B strains were recovered from the cases of septicemia hemorrhagica in Asia and some parts of Europe. Type E has only been reported from septicemia hemorrhagica cases in Central Africa (Carter, 1961; Perreau, 1961). In the northern hemisphere and in countries with a temperate climate, type A has been found to predominate. Most of the Pasteurella strains from cattle in Canada.and the United States were associated with pneumonia (Carter, 1957b). Consequently it was recommended that P. multocida type A and P. hemolytica be included in bacterins and antisera for the prevention of shipping fever in Canada. The latter species of Pasteurella was found to be an important organism in the l6 etiology of shipping fever in Canada (Carter, 1954, 1956). Consistent with the previous recommendations of Bain (l954a) and Hudson (1954), Carter also recommended that type B or Roberts' type I should be employed for the production of immunizing agents for the prevention of epizootic septicemia hemorrhagica where it usually occurs in Asia and Africa. Indirect hemagglutination tests and mouse protection tests have been carried out by Perreau at al. (1964) to determine if antibodies were present in 411 Zebu cattle in the Republic of Chad, Africa. The examination of 82 samples showed specific antibodies to type E capsular antigen indicating that septicemia hemorrhagica was enzootic in that country. Namioka and Bruner (1963) employed the IHA tests and tube agglutina- tion procedures to identify capsular and somatic antigens of 23 bovine strains. Their results are summarized in Table 4. Table 4. Serotypes of bovine strains of Pasteurella multocida isolated from several countries No. of Associated Capsular strains Origin disease Type 0 Group Serotype 1 Burma Sepsis A 7 7:A Chad (Africa) Septicemia - 6 6:- hemorrhagica 4 Egypt Septicemia B 6 6:B hemorrhagica 3 Philippines Sepsis A 7 7:A 3 U.S.A. Pneumonia (l) A l 1:A Pneumonia (1) D - -:D ? (1) - 6 6:- 10 Vietnam Sepsis (2) - 7 7:- Sepsis (1) A 7 7:A Septicemia - 6 6:- hemorrhagica (7) 17 In other studies Murata et al. (1964) pointed out the importance of 0 antigen as one of the determining factors in the antigenicity of P. multocida. Further studies of Namioka and Murata (1964), employing absorption tests, indicated that 2 kinds of 0 group could be identified from Carter's type B, viz., 6 and 11. It has been shown that strain 11:B, isolated from a cattle wound in Australia, did not produce septi- cemia hemorrhagica. The finding that strain Bunia II, isolated from the cases of septicemia hemorrhagica in Congo, Africa, belonged to 0 group 6 (Carter's type E), supported their postulate. MATERIALS AND METHODS Typing of Bovine Strains of P. multocida Strains of P. multocida Forty bovine strains mainly from midwestern United States were examined serologically. The numbers identifying the strains, the clinical source, and the geographic origin are presented in Table 5.‘ After original isolation they were maintained on Stock Culture agar (Difco). Cultures were transferred every 4 or 5 months to fresh stock culture media. aegis Blood (5—6% ox or human group O) agar was the most frequently used. Serum tryptose agar was used for the identification of cultural variants. Drying out of plates was prevented by storing them in the refrigerator in sealed plastic bags. Brain heart infusion (BHI) (Difco) broth was used in the acriflavine test. Preparation of Antisera Two rabbits were used to produce antisera for each strain of known serotype. Each usually received 10 intravenous injections of 0.25% formalin-killed vaccine with dosage starting at 0.5 ml. and increasing by increments to 5.0 ml. To prepare the vaccine, 18-24 hour cultures were harvested from blood agar media by washing off with 10.0 ml. of 18 19 Table 5. Source of the bovine strains of Pasteurella multocida Strain No. Associated Disease Geographic Origin/Donor 702 Pneumonia Pitman MOOre, New Jersey B- 175-69 Pneumonia Michigan, MSU B-1085-69 Bacteremia Michigan, MSU B-1087-69 Pneumonia Michigan, MSU B-1149-69 Bacteremia Michigan, MSU B-1112-7l Chronic pneumonia Michigan, MSU B-1130-7l Pneumonia Michigan, MSU 704 Pneumonia Pitman Moore, New Jersey M-l Pneumonia Minnesota, Dr. Johnson. M-2 Pneumonia Minnesota, Dr. Johnson. M-3 Shipping fever Minnesota, Dr. Johnson. M-4 Pneumonia (?) Minnesota, Dr. Johnson M-5 Pneumonia Minnesota, Dr. Johnson M-6 Pneumonia Minnesota, Dr. Johnson M-7 Pneumonia Minnesota, Dr. Johnson M28 Pneumonia Minnesota, Dr. Johnson M-9 Pneumonia Minnesota, Dr. Johnson MrlO Upper respiratory Minnesota, Dr. Johnson infection M911 Upper respiratory Minnesota, Dr. Johnson. infection M-lZ Upper respiratory Minnesota, Dr. Johnson infection Mrl3 Upper respiratory Minnesota, Dr. Johnson infection M-l4 Upper respiratory Minnesota, Dr. Johnson infection B- 218-71 Pneumonia Michigan, MSU B- 365-71 Upper respiratory Michigan, MSU infection B-1230-71 Upper respiratory Michigan, MSU infection B- 568-69 Pneumonia, nephritis Michigan, MSU 154 Pneumonia (7) Michigan, MSU 20 Table 5 (cont'd.) Strain No. Associated Disease Geographic Origin/Donor 151 Pneumonia (?) Michigan, MSU B- 220-68 Pneumonia Michigan, MSU 341406-71 Pneumonia Michigan, MSU B—l359-71 Pneumonia Michigan, MSU B-l363-7l Pneumonia Michigan, MSU B-l476-71 Pneumonia Michigan, MSU BTfi 20-72 Pneumonia Michigan, MSU BT-333-72 Pneumonia. Michigan, MSU- BT-372-72 Mastitis (?) Michigan, MSU B-lO46-69 Pneumonia Michigan, MSU B-1048-69 Bacteremia Michigan, MSU L-15743 Pneumonia Indiana, Lilly Labs L-700 Upper respiratory Indiana, Lilly Labs infection sterile physiological saline solution (PSS). The suspension.was centri— fuged at 2852 x g for 30 minutes after which the sediment was.resuspended with sterile PSS to make a total volume of 40.0-50.0 ml. Formalin was. added to give a final concentration of 0.25%. After overnight incubation at 37 C, a sterility test was performed by placing 1 or 2 drops of the bacterial suspension into a tube of BHI semisolid broth. Injections were made at 4-day intervals, and 4 days after the last injection the rabbit was exsanguinated by cardiac puncture. The anti- sera were inactivated at 56 C for 30 minutes and preserved by adding 1% of a 1% solution of sodium ethylmercurithiosalicylate.* All antisera were stored in the freezer until used. *“ Thiomersal, K & K Laboratories, Plainview, N.Y. 21 Performance of Indirect Hemagglutination Test The procedure used for the indirect hemagglutination (IHA) test was. that of Carter (1955, 1972b). Strains of P. multocida were grown on blood agar for 18 to 24 hours, then washed off with 3.0 ml. of phosphate buffered saline (PBS) pH 6.0. To the suspension was added 1.0 m1. of PBS le6.0 containing 15 National Formulary (N.F.) units (50 viscosity reducing units) of hyaluronidase.* The suspension was then incubated in a waterbath at 37 C for 4 hours, to allow maximum release of the capsular material (antigen). The bacteria were removed by centrifuga- tion at 2852 x g for 30 minutes and the supernatant was transferred into another tube. To this was added 0.1 m1. washed human group 0 red blood cells. Treated red blood cells were separated by centrifugation, washed 3 times with PSS and resuspended in 20.0 ml. of PSS to make a 0.5% suspension. Twofold dilutions, 1:10 through 1:320 were made in Kahn tubes from the known capsular type A and D antisera. To each tube containing 0.25 ‘m1.'diluted antiserum was added 0.25 ml. of the treated red blood cells suspension. The tubes were gently shaken and allowed to stand at room temperature. Readings were usually made twice; the first at 2 hours after the addition of the red blood cells and the second after standing overnight. A reaction was considered positive if agglutination occurred in the form of clumps observable in the bottom of the tube after light agitation or the blood cell "button" failed to run when the tubes were tilted. The degree of positiveness was evaluated from one plus to four depending on the "tightness" of the clumping of the red blood cells. Control tubes consisted of the same twofold dilutions_of antisera to * Haglodase, Haver—Lockhart Laboratories, Shawnee, Kansas 66201. 22 which were added 0.5% suspension of untreated red blood cells. Those antisera that clumped untreated red cells were adsorbed with washed human group 0 red blood cells. Rapid Agglutination Test Agglutination tests were conducted on a standard plate of the kind usually used to detect the presence of antibodies to Brucella abortus. The bacteria remaining after hyaluronidase treatment for the IHA test were used in the rapid agglutination test. First they were washed with PSS then suspended in 2.0 m1. of 0.3% formol PBS, pH 7.2. One dr0p of each dilution of antiserum (1:10 to 1:320) of known serotype was placed on the squares of the plate. To each of these, 1 drop of antigen was added. Mixing was accomplished by stirring with an applicator stick. The reading of reactions was accelerated by rotating the plate gently. Positive reactions occurred within approximately 2 minutes and in some instances delayed reactions were observed up to 5 minutes. Reactions occurring after 5 minutes were considered false or questionable. Acriflavine Test The method employed was a modification of the slide acriflavine test described by Braun and Bonestell (1947) and Carter (1957). It differed in the amount of bacteria employed and the fact that it was carried out in a tube instead of on a microscope slide. Each strain examined was grown in 3.0 ml. of BHI broth at 37 C for 24 hours. The culture was centrifuged and 2.0 ml. of the supernatant was removed. The bacteria were then resuspended in the remaining 1.0 ml. of medium. 23 Acriflavine solution (1:1,000) was prepared by dissolving 100.0 mg. of acriflavine* in 100.0 ml. of distilled water. The solution was prepared each week and stored in a brown bottle. The test was conducted by adding 0.5 ml. of acriflavine solution (1:1,000) to a tube containing 0.5 ml. of concentrated bacteria. After mixing, a reading was made in approximately 5 minutes. Type D strains produced coarse granular clumps which settled within 2 or 3 minutes. The cells obtained from mucoid strains would remain in suspension. Smooth (SR) and rough (R) strains would flocculate and settle to the bottom of the tube giving a gel-like appearance. If coarse clumping occurred the acriflavine solution above the settled-out precipitate was a clear yellow color closely resembling the original color. Detection of P. multocida Antibodies in Bovine Sera Sixty samples of blood taken before vaccination and 50 samples taken 2 months postvaccination were collected from the Beef Cattle Research Center (BCRC) of Michigan State University,_LakeCity, Michigan. Vaccination was made by giving a single subcutaneous ** injection of Pasteurella bacterin. Preparation of the Serum Serum was decanted from the clotted blood and centrifuged to remove red blood cells. Inactivation was carried out at 56 C for 30 minutes. All sera were adsorbed with group 0 red blood cells as follows: 0.3 ml. of washed erythrocytes were suspended in a tube containing 1.7 ml. of formol saline and 0.5 m1. of serum. The suspension was stored at 4 C * Acriflavine Neutral, N.F., Allied Chemical Corp., New York 6, N.Y. ** Septobac, Fort Dodge Laboratories, Inc., Fort Dodge, Iowa. 24 overnight. The following day the adsorbed serum was recovered from the red cells by centrifugation. Controls were provided to detect the agglutination of untreated red blood cells. Indirect Hemagglutination (IHA) Test The IHA test was performed as previously described using strain B-l476-71 as source of capsular antigen. Indirect Hemagglutination Test Employing a Microtiter Technique* Basically this procedure was the same as the conventional tube indirect hemagglutination test, except microvolumes were used. The tests were conducted in a "U" plate with 96 wells using 25 microliter pipette drOppers to add the saline and the antigens. Single micro- diluters (25 microliter) were used to prepare twofold dilutions of sera. The system required only one tenth the amount of antigen and antiserum employed in the conventional IHA test. Drying out of the plate during the tests was prevented by covering it with damp paper. The tests were read as described for the conventional IHA tests. Rapid Agglutination Test This was performed as those for the serotyping of Pasteurella strains, using strain B-702 as the source of antigen. * Cooke Engineering Company, Alexandria, Va. 22314. RESULTS Part 1 Antibody Titers of Rabbit Antisera Antisera Employed for the IHA Test. Because only type A and type D strains of P. multocida have been reported from cattle in North America only these type sera were employed for the capsular typing. The diffi- culty of obtaining high titer type A antiserum has been mentioned by Carter (1958). In this study similar difficulty was experienced in obtaining potent type D sera. The capsular typing sera employed, their origin and IHA titer are listed in Table 6. Table 6. Titers of antisera employed in the IHA test .WV— r. a. Indirect hemagglu- tinationItiter Strain Origin Capsular Type. (Homologous Antigen) X273-2 Chicken. A 320 P-48-71 Porcine A 320 D1* ? D 160** Kobe-6 Porcine D 80 * Serum kindly supplied by Dr. P. Perreau, France. ** - Employing Strain Kobe-6 as antigen. 25 26 Types B and E antisera were also available but none of the strains examined was identified as belonging to these capsular categories. Antisera Employed for RA Test. The antisera prepared, their identifi- cation and titers obtained with homologous bacterial suspensions are listed in Table 7. Pairs of rabbits were inoculated with 7 of the strains listed in Table 8. In all of the pairs except 1 the titers were of comparative strength. The only antisera that reacted in low titer with all antigens were those prepared from strain B-1130-7l. Table 7. Typing sera employed for the rapid agglutination test Somatic Group Strain Origin (Tentative) RA Test Titer 3397-1 Porcine 1a 160 M-6 Bovine 1a,b 320 P-48-7l Porcine IC 160 Capsular Types Determined by Indirect Hemagglutination Test. The results of subjecting all 40 bovine strains of P. multocida to the IHA test are summarized in Table 9. All of the_strains were typable. Thirty-six strains (90%) were type A and 4 (10%) were type D. The degree of posi- tiveness ranged from 1+ to 4+ and presumably dependefl upon the amount of_ antigen released by each strain. Results of Examination of Strains with the Rapid Agglutination Test. Based on the results of the agglutination tests between the antisera and homologous antigens listed in Table 8, sera representing what were FLEHII Essex .camuom mauouom « 03 own Q.» 28 own cum omm omm own I 8H 0.» I I own own 03 o: I :7me I I I I I I I I I I I I I I I I I I I rolmooM I Own ooa omm omm omm own own omm I I ON I I cmm own I I as «mmmm I I I I I I I I I I I I I I I I I as om NmINNmIHm I I I I I I I I ow I I I I I I I I I I «Tom Jam ow cum ow cmm omm can own omm omm oe cs o.» as I own 36 as on: 0.» :Iong I own I 03 cs 03 o3 own 03 I I cm I I omm cum I O.» on modem—Tm I own I owm om on 00.... omm om I cc 0.» I I 9: 03 I I I :Iomznm I owm om owm ooa omm own can own I I I I I own omm I I om 0|: om omm ow omm omm omm on omm omm I on: ow I I own omm can own ow mix I own or: own can own on omm omm oc I o.» om om omm own on: 9: omm NE 8 m m M M "nu ma mu 4. ma an commutes d _ _ _ _ . T. T. T. .L .L T. . x s I e s w w w w n n .4. m .n m m. mm W. “w m” N“ w“ “W MW 0, o, ,6 ,6 no no 0, o, C. C. .6 . a I. o, _ _ _ . _ _ _ _ _ _ _ _ . . I. _ _ _ I. I. I. I. I. I. 9 9 I. I. .6 T. 7o T. .L 0, TL o, 76 7s 7c .6 J. 4. ,w ,w .% a. ,6 no u. ho F. 7V T. 7y .L .6 T. asumm ummu mowummfiusawwm names may as commando mnowoaoaon one mummwuam mo mumuHH .w pansy 28 Table 9. Capsular types of bovine strains of P. multocida determined by IHA test Strain No. Capsular or K Antigen. 702 B- 175-69 B-1085-69 B-1087-69 B-1149-69 B-1112-7l B-ll30-71 704 Mél M-2 M-3 M-4 M-5 M-6 M—7 M—8 M-9 M-lO M211 M-12 M-13 M~l4 B- 218-71 B- 365-71 B-1230-7l B- 568-69 154 151 B- 220-68 B-1406-7l B-1359F71 B-1363-7l B-1476-7l BT- 20-72 BT-333-72 BT-372-72 B-1046—69 B-1048-69 L-15743 L-7OO >>>>U>U>>>>>>>>fi>>v>>>>>>>>b>>>>U>>>>>>>>> 29 considered the principal 0 group categories were selected for the sero- logic examination of the remaining strains. The results are presented in Table 10 and, based on these results, tentative O or somatic antigenic identification was assigned. These along with the capsular type are listed in Table 11. Part 2 Occurrence of Antipasteurella Antibodies in Bovine Sera Antibodies Measured by the IHA Test: Prevaccination. Examination of the sera collected from the Beef Cattle Research Center (BCRC) prior to vaccination employing IHA tests using strain B-1476-7l as antigen. indicated that 4 of the 60 samples possessed low titers of antibody. to P. multocidb type A. Examination employing the microtiter procedure provided results which paralleled those of the former procedure. The- results obtained with both procedures are presented in Tables 12 and 13. Antibodies Measured by the IHA Test: Postvaccination. Of the 50 serum samples made available, 25 were derived from cattle that had not received Pasteurella bacterin. Those that had not been vaccinated with the Pasteurella organisms were vaccinated with "IBR"* and "Nasalgen-P"** vaccines, or "Respacine 2"* vaccine. Those receiving the PasteureZZa bacterin had been injected simultaneously either with."IBR" and. "NasalgenéF'vaccines, or with "Respacine 2" vaccine. All samples except 1 possessed low to high titers of antibody to P. multocida type A. The microtiter procedure was carried out and the * Diamond Laboratories, Des Moines, Iowa. ** ‘ Jensen-Salsbery Laboratories, Kansas City, Mo. 64141. 30 Table 10. Results of rapid agglutination test with selected 0 group antisera Sera Agglutination Titer 3397-1 M-6 P-48-71 Somatic Antigenic Antigen‘ (a) (b) (c) Identification 702 160 320 - 1a,b B- 175-69 - 160 - lb. B-1085-69 20 320 - 1a,b B-1087-69 - 320 - lb B-1149-69 20 320 - 1a,b B-1112-71 20 160 - 1a,b B-1130-7l - 160 - 1b. 704 - 160 - 1b M-l 20 320 - 1a,b M-Zr — - - ? M+3 40 320 - 1a,b M-4 20 160 - 1a,b M-S 80 320 80 1a,b,c M96 20 320 - 1a,b M-7 - - - ? Mr8 160 320 160 1a,b,c M-9 80 160 - 1a,b M910 - - - Autoagglutinated M-11 80 320 - 1a,b M-12 80 320 20 1a,b,c M-13 40 320 - 1a,b. M914, 160 320 80 1a,b,c B- 218-71 - 320 - 1b B- 365-71 80 320 160 1a,b,c B-1230-71 40 320 - la,b' B- 568-69’ - 320 - lb 154 40 320 160 1a,b,c 151 - 320 - 1b B- 220-68 - - - ? B-1406-71 - - - Autoagglutinated B-1359-71 - - - Autoagglutinated B-1363-71 40 320 20 1a,b,c B-1476-71 80 320 80 1a,b,c BT- 20-72 - - - ? BT—333—72 - - - Autoagglutinated BT-372-72 - - - ? 361046-69 - - - ? B-1048r69 - 320 - 1b L-15743 80 320 - 1a,b L4700 160 320 160 1a,b,c 31 Table 11. Serologic identification of bovine strains of PasteureZZa multocidb 0 or Somatic Antigens K or Capsular Serotype Strain No. (Tentative) Antigen (Tentative) 702 1a,b A 1a,b :A B- 175-69 1b A 1b :A 8-1085-69 1a,b A 1a,b :A 8-1087-69 lb A lb :A B-1149-69 1a,b,c A 1a,b,c:A B—1112-7l 1a,b A 1a,b :A B-ll30-71 lb A lb :A 704 lb A lb :A Mrl 1a,b A 1a,b :A MEZ - D - :D M-3 1a,b A 1a,b :A M-4 1a,b A 1a,b :A M—S 1a,b,c A 1a,b,c:A M—6 1a,b A 1a,b :A Mr7 — D - :D M28 1a,b,c A 1a,b,c:A M99 1a,b A 1a,b :A MrlO - A - :A Mell 1a,b A 1a,b :A M—12 1a,b,c A 1a,b,c:A M-13 1a,b‘ A 1a,b :A MélA 1a,b,c A 1a,b,c:A B- 218-71 1b A lb :A B- 365-71 1a,b,c A 1a,b,c:A B-1230-71. 1a,b A 1a,b :A B- $68-69 lb A 1b :A 154 1a,b,c A 1a,b,c:A 151 lb A lb ~ :A B-A220-68 - A ' - :A 341406-71 - A - :A B-1359-7l - A - :A B-1363—71 1a,b,c A 1a,b,c:A B-1476-7l 1a,b,c A 1a,b,c:A BT- 20-72 - D - :D BT-333-72 - A - :A BT—372-72 — D - :D B-1046-69 - A - :A B-1048-69 lb A lb :A L-15743 1a,b A 1a,b :A L-700 1a,b,c A 1a,b,c:A 32 Table 12. Pasteurella antibody titers employing IHA test in cattle prior to vaccination Reciprocals of Final Serum Dilution Sample No. 20 40 80 160 320 640 65 61 22 15 8 1 135 129 93 88' 80 78 124 42 158 156 143 142 54 46 105 29 115 113 354 372 304 288 337 388 257 201 269 218 324 235 260 243 209 226 361 344 I++I I I I I +IIIIIIIIIIIIIIIIIIIIIIIIIIIiIIIII-F‘I'IIIIII I I I I I 33 Table 12 (cont'd.) Reciprocals of Final Serum Dilution; Sample No. 20 40 80 160 320 640 310 - - - - - - 276 - - - - - - 293 — - - - - - 327 - — - - - - 378 - - - - - - 395 - - - - - - 267 - - - - - - 251 - - - - - - 234 - - - - - - 217 - - - - - - 334 - - - - - - 317 - - - - - - 300 - - - - - - 283 - - - - - - 385 - - - - - - 400 - - - - - - 368 - - - - - - 351 - - ~ - - - - 34 Table 13. PasteureZZa antibody.titers employing IHA test using micro- titer-technique (strain B-l476-7l as antigen) in cattle prior to vaccination Reciprocals of Final Serum Dilution Sample No. 20 40' 80 160 320 640 1280 65 - - — - - - - 61 - - - - - - - - 22 - - - - - - - 15 - - - - - - - g - - - - - - - 1 - - - _ - - - 135 + + + + - - - 129 + + + - - - - 93 - - . - - - — - 88 - - - - - - - - 78 - - - - - - ' - 124 - - - - - - - 42 + + + + - - - 158 - - - - - - - 156 - - - - - - - 143 - - - - - - .- 142 - - - - - - - 54 - - - - - - - 46 - - - - - - - 105 - - - - - -' - 29 - - - - - ' - - 115 - - - - - - - 113 - - - - - - - 354 - - - - - - - 372 - - - ‘ - - - - 304 - - - - - - - 288 -4 - - - - - - 337 - ’ - - - - - - 388 - - - - - - - 257 - - - - - - - 201 - - - - - . - - 269 - - - - - - - 218 - - - - - - - 324 - - - - - - - 235 - - - - - - - 260 - - - - - - - 243 - - - - - - - 209 - - - - - - - 226 - - - - - - - 361 - - - - - - - 344 + + + + + - - 8O - - - - - - - 35 Table 13 (cont'd.) Reciprocals of Final Serum Dilution Sample No. 20 40 80 160 320 640 1280 310 - - - - - - - 276 - - - - - - — 293 - - - - - - - 327 - - - - - - - 378 - - - - - - - 395 - - - - - - - 267 - - - - - - - 251 - - - - - - - 234 - - - - - - - 217 - - - - - - - 334 - - - - - - - 317 - - - - - - - 300 - - - - - - - 283 - - - - - - - ‘ 385 - - - - - - - 400 -. - - - - - - - 368 - - - . - - - - 351 - - - - - - - 36 results were comparable to those obtained with the conventional or tube IHA test. Serologic examination employing strain Kobe-6 (capsule type D) as source of antigen in the IHA test did not result in any positive reactions. The results using both procedures are presented in Tables 14, 15, 16, and 17. Antibodies Measured by the RapidgAgglutination Test: Prevaccination. Employing strain B-702 as the source of organisms, the RA test was. carried out on 60 prevaccination serum samples to determine if they had somatic antibody of 0 group 1a,b. Seven samples (12%) possessed moderate titers of antibody. The results are presented in Table 18. Antibodies Measured by the Rapid Agglutination Test:~ Postvaccination. Nine of the 25 serum samples obtained from animals that had not been vaccinated with Pasteurella bacterin possessed low to moderate amounts of antibody. Nine of the 25 serum samples obtained from animals that had been vaccinated with PasteureZZa bacterin also possessed low to- high amounts of antibody. The results are presented in Tables 19 and 20. Antibodies to P. hevaytica Type I Measured bygthe IHA and the Agglutina- tion Tests. Both groups of sera, pre- and postvaccination, were examined for capsular and somatic antibodies to P. hemolytica type I. The tube agglutination and the IHA tests were performed according to the methods. of Biberstein et al. (1961) and Carter (1955). Except sample no. 276 (postvaccination), all samples gave negative results to the above tests. Sample no. 276 was positive (1:80) in Biberstein's agglutination test. The IHA tests with P. hemolytica were not altogether satisfactory in that no known positive serum was available to show that red cells had been adequately coated with specific antigen. 37 Table 14. Pasteurella antibody titers (IHA test) in sera collected from control animals 2 months after the initial test using strain B-1476-71 as antigen Reciprocals of Final Serum Dilution Remarks Sample No. 20 40 80 160 320 640 (Degree of Titer) 22 + + + - - - moderate 15 + + i - - - moderate . 93 + +' + + - - moderate 124 +' + + .i_ - - moderate- 156 + + i - - - moderate 143 + + + J: - - moderate 142 -H-l- +++ ++ + + - high 54 + + + + - - moderate 115 ++ ++ + + + + high ‘304 + + + - - - moderate 388 + + + .i. - - moderate 257 ++ ++ + .i_ - - moderate 235 + + + :l-_ ~ - - moderate 260 ++' ++ + + - - moderate 243 -H- ++ + + i - high 310 + + i; - - - moderate 327 + + + - - - moderate 378 + + + i - - moderate 395 +_ + + - - - moderate 267 + + - - - - low 251 ++ ++ + + - - moderate 334 -H- -H- + I + _-l_-_ - high 317 + + + .i_ - - moderate 400 +-H- , ++ ++ + + + high 312 ++' ++ + + - - moderate 38 Table 15. Pasteurella antibody titers (IHA test) in sera collected from vaccinated animals 2 months after the initial test using strain B-1476-7l as antigen. Reciprocals of Final Serum Dilution Remarks Sample No. 20 40 80‘ 160 320 640 (Degree of Titer) 65 ++ + + + + - high 61 ++ + + - - - moderate 8 ++- ++ + + - - moderate l + + + + - - moderate 80 - - - - - - - 78 ++ + + - - - moderate 105 -H-I- ++ + + + + high 29 +*+- ++' + + - - moderate 113 ++- ++ + + - - moderate 354 + + + + - - moderate 337 -H-l- -H- ++ + + + high 201 + + i . - - - moderate 269 + + + i - - moderate 218 + + - - - - low 209 -H- -H- + + i - high 226 + + + + - - moderate 361 + + - - - - low 344 -I-I- -H- ++ + - high 276 + + + + - - moderate 217 + + - - - - low 300 -H+ ++ + + + - high 283 ++-H- ++ + + + i high 368 + + + - - - moderate 351 + + + - - - moderate 213 ++ ++- + + - - moderate 39 Table 16. Pasteurella antibody titers (IHA test: microtiter technique) in sera collected from control animals 2 months after the initial test using strain B-l476-7l as antigen Reciprocals of Final Serum Dilutionpr Remarks Sample No. 20 40 80 160 320 640 1280 (Degree of Titer) 22 + + + - - - - moderate 15 + + + - - - - moderate 93 + + + - - - - moderate 124 +' + + + - - - moderate 156 + + + - - - - moderate 143 + + + - - - moderate 142 + + + + + + + high 54 + + + - - - - moderate 115 + + + - - - - moderate 304 + + + + - - high 388 + + + - - - - moderate 257 + + - - - - - low 235 + + + + - - - moderate 260 + + + - - - - moderate 243 + +. + + - - - moderate 310 + + + + - - - moderate. 327 + + + + - - - moderate 378 + + + + - - - moderate 395 +- + + - - - — moderate 267 + + + — - - - moderate 251 + . + + - - - - moderate 334 + + + - — - - moderate. 317 + + + - - - - moderate 400 + + + + + + + high . 312 + + + - - - - moderate 40 Table 17. Pasteurella antibody (IHA test: microtiter technique) in sera collected from vaccinated animals 2 months after the initial test using strain B-l476-71 as antigen. Reciprocals of Final Serum.Dilution Remarks Sample No. 20 40 80 160 320 640 1280 (Degree of Titer) 65 + + + + + + - high 61 + + + - - - - moderate 8 + + + - - - - moderate l + + + - - - - moderate 80 - - - - - - — - 78 + + + - - - - moderate 105 + - + + + + - - high« 29 + + + - — - - moderate 113 + + + + + - - high 354 + + + + + - - high 337 + + + + + - - high 201 + + - - - - - low 269 + + + + - - - moderate 218 + + + - - - - moderate. 209 + + + + + - - high 226 + + + - - - - moderate 361 + + + + - - - moderate. 344 + + - — - - - low 276 + + + - - - - moderate 217 + + + - - - - moderate. 300 + + + - - - - ‘moderate 283 + + + + + - - high 368 + + -+- + - - - moderate. 351 + + + + - - - moderate 213 + + + - - - — moderate 41 Table 18. Pasteurella antibody titers employing rapid agglutination test in cattle prior to vaccination, using strain B-702 (0 group la,b) as antigen Reciprocals of Final Serum Dilution Sample. No. 20 4o 80 160 320 640 65 - - - - - - 61 + + + - - - 22 + + + — - .- 15 + + + - - - 3 - _ - - - _ 1 _ _ - _ - - 135 - - - - - - 129 - - - - - - 93 - - - - - - 88 - - - - - - 80 - - - - - - 78 - - - - - - 124 - - - - - - 42 + + + - - - 158 - - - - - - 156 + + + - - - 143 - - - - - - 142 - - - - - - 54 - - - - - - 46 + + - - - - 105 - - - - - - 29 - - - - - - 115 - - - - - - 113 + + + - - - 354 - - - - - - 372 - - - - - - 304 - - - - - - - 288 - - - - - - 337 - - - - - - 388 - - - - - - 257 - - - - - - 201 - ' - - - - - 269 - - - — - - 218 - - - - - - 324 - - - - - - 235 - - - - - - 26o - - - - - - 243 - - - - - - 209 - - - - - - 226 - - - - - - 42 Table 18 (cont'd.) Reciprocals of Final Serum Dilution Sample No. 20 4o 80 160 320 361 - - - - - 344 — - - _ _ 310 - - - - - 276 - - - - - 293 - - - - - 327 - - - - - 378 - - - - - 395 - - - - - 267 - - - - - 251 - - - - - 234 - - - - - 217 - - - - - 334 - - - - - 317 - - - - - 300 - - - - - 243 - - - - - 385 - - - - - 400 - - - - - 368 - - - - - 351 - - - - - 43 Table 19. PasteureZZa antibody titers (RA test) in sera collected from control animals 2 months after the initial test using strain B-702 as antigen: Recriprocals of Final Serum.Dilution Remarks Sample No. 20 40 80 160 320 640 (Degree of Titer) 22 15 93 124. 156 143 142 54 115 304 388 257 235 260 243 310‘ 327 378 395 267 251 334 - - - - - - - 317 - - - -‘ - - - 400 312 - -- - - - - - + I I I moderate - - - moderate + - - moderate - - - low + - - moderate I+l+|+| - -. - - low - -. - - low + I I I I I I I + + I I i + + I + I I I + + I I I I I I I +-+ I I I + +4I + I I I + + + l I I I '2‘ 44 Table 20. Pasteurella antibody titers (RA test) in sera collected from vaccinated animals 2 months after the initial test using strain B-702 as antigen. Reciprocals of Final Serum Dilution Remarks Sample No. 20 4O 80 160 320 640 (Degree of Titer) 65 + + + - - - moderate 61 - - - - - - - g - - _ - - - - l + + - - - moderate 80 - - - - - - - 78' - - - - - - - 105 - - - - - - - 29 + + - - - - low 113 + + - - - - low 354 - - - - - - low 337 - - - - - - - 201 -‘ - - - - - - 269 - - - - - - - 218 - - - - - - - 209 + + + + + - high 226 - - - - - - - 361 - - - - - - - 344 - - - - - - - 276 + + - - - - low 217 — - - - - - - 300 + + + - - - moderate- 283 + + + - - - moderate 368 + + - -, - - low 351, - - - - - - - 213 1".“ . 71:7. .11 DISCUSSION Part.l Mostof the bovine strains of P. multocidb studied were associated with the pneumonic type of pasteurellosis.' Likewise the earlier study of Carter (1957b) involved principally bovine strains from cases of pneumonia. Ninety percent of the strains examined were found to belong to capsular type A, and 10% to type D. These figures indicate that in the areas from which the strains were isolated the majority possessed capsular type A. In the IHA tests, varying.degrees of positive reac- tions were evident with the same typing sera indicating differences in amounts of capsular antigen. Strains that possessed large, moist, and mucoid colonies usually gave strong positive reactions, while small, smooth, "dry" colonies consisting of poorly capsulated organisms. resulted in weak reactions.. To increase the capsulation of organisms some strains were passed in embryonated chicken eggs and in mice. Although strains were usually pathogenic, typability could not always be restored. While performing the RA tests, it was noted that some strains tended to autoagglutinate. In such instances, efforts were made to obtain stable antigens by passage of strains in eggs and mice. This procedure was not usually successful in restoring antigenic stability. It was noted that some.strains were variable in their stability in that 45 46 one antigen preparation would be agglutinable but a later culture prepa- ration might be.inagglutinable. Some of the difficulty in obtaining specific anti-O sera may have been due to "normal" antipasteurella antibodies present in the rabbits prior‘to.antigen inoculations. It is well known that P. multocidb is widely distributed among rabbits. Low "natural" titers could be increased as a result of repeated injection of killed P. multocidh- This is probably explainable on the basis of what immunologists call the "doctrine of original antigenic sin" (Davis et al., 1969). To. determine if this hypothesis was valid, at the conclusion of this study sera were collected from 12 unimmunized rabbits from the same.source as the rabbits used in the investigation. These were examined by the RA test using strains B-1476-7l, M—6, and P-180-71 as sources of organisms. The results which confirm the hypothesis are summarized in Table Arl (Appendix). It is of interest that Namioka and his associates described an outbreak of pasteurellosis in rabbits due to serotype.l:A (Sate et al., 1967). It is well known from the work of Namioka and Murata (1961b) that the 0 antigen composition of P. multocidh is very complex. In order to obtain "specific factor sera" they had to absorb all of their sera with large quantities of heterologous organisms. ,Carter-(l972c), in a study of avian strains, noted that there was less need for absorption of sera with his RA procedure, although he found it necessary to absorb some anti-Orsera. Considerable cross reactions were observed among the strains of this study (see Table 8). Selected sera were absorbed with the heterolo- gous strains to determine if the absorbed sera still agglutinated the homologous strain. If they did it was.conc1uded that the cross reaction 47 was due to a "secondary" determinant. If there was no reaction with the homologous strain.it was concluded that the homologous strain and the strain giving the lesser reaction were antigenically similar. Based upon the absorptions and the pattern of the cross reactions it was concluded that strains usually possessed more than one antigenic determinant (Table A-2, Appendix). Subgroups a, b, and c were tenta- tively assigned to 0 group 1 and strains were thus designated by 1 followed by the letters indicating subgroups. The presence of different 9 subgroups among 0 group 1 has been observed by Namioka and Murata ! i ! | Type D strains BT-20-72, BT—372—72, and Kobe-6 (porcine) elicited high titers of antipasteurella antibodies in rabbits (Table 8). However, k these antisera did not agglutinate the homologous antigens in the RA tests. This was probably due to the fact that type D strains, unlike type A strains, are not rendered agglutinable by hyaluronidase. Perhaps the acid treatment employed by Namioka and Murata would have made the suspensions agglutinable. The reactions obtained with decapsulated type A strains in the RA test indicated a close similarity of the 0 groups of type A and D strains. However, it was thought advisable not to assign 0 group identities to the type D strains at this time. Of the ten strains that were not satisfactorily identified, 4 were type D, 4 were autoagglutinable and 2 were type A with unknown 0 group: composition.‘ They might belong to different subgroups of 0 group 1, or to the other 0 groups. Based on the results of these studies, bacterins recommended for the prevention of bovine pasteurellosis in the United States should be prepared from strains representing the serologic varieties identified in this investigation. 48 Part 2 The Pasteurella bacterin used in this study was prepared from P. multocidd types A and D (50%), and P. hemolytica (50%). According to the direction for use given by the manufacturer, this product should be injected twice with a.5- to 7-day interval. As has been mentioned earlier, animals receiving Pasteurella bacterin were injected simul-- taneosuly.either with "IBR" and "Nasalgen—P" or with "Respacine‘2" vaccines. A single dose of PasteureZZa bacterin was used to vaccinate. H the animals in this study.’ Of 60 serum samples collected prior to vaccination, 4 were found to possess low titers of antibody to P. multocidh type A. After the vaccination, antibody to type A was found in all 25 animals that had. 1—— -_.—_— .4 - ._. _ not been injected with the bacterin. Of those (25) vaccinated with the bacterin, 24 showed low to high titers of antibody. No significant difference was observed in the antibody level to type A capsular sub- stance in both groups of animals. Since animals not receiving the-bacterin showed appreciable anti- body titers, it seems likely that these titers of antibodies resulted from exposure to Pasteurella organisms. In support of this thesis is the fact that after vaccination the cattle being studied sustained a mild outbreak of respiratory infection that was called shipping fever. clinically. More than 28% of animals from all groups displayed clinical signs of disease. Aside from the antibody to P. multocida type A, antibodies to type- D and to P. hewvlytica were not found in this study, although in the case of P. hemolytica it was not certain that the test strain.was- adequately capsulated. 49 Antibody to somatic antigen group 1a,b was found in 4 of 60 serum samples collected prior to vaccination. Of 25 serum samples obtained from animals that did not receive the PasteureZZa bacterin, 9 showed low to moderate titers of antibody to 0 group 1a,b. Similar figures were obtained with the samples derived from animals vaccinated with the bacterin. There was no significant difference in the antibody titers between the 2 groups of animals. One can only conclude that there is no evidence from this investigation to indicate that a single dose of Pasteurella bacterin produced a significant humoral immune response. As for the serologic techniques employed, it was shown that the microtiter procedure may be used effectively in the IHA test. The advantage of this procedure lies in the smaller amounts of antigen and antiserum required as compared with that needed for the conventional method; however, the conventional IHA test is easier to read than microtiter procedure. SUMMARY The indirect hemagglutination (IRA) and rapid agglutination (RA) tests-were employed to study the serologic and antigenic nature of 40 bovine strains of P. multocida isolated from cattle with pneumonia in several areas of midwestern United States. Colonial characteristics and the acriflavine test were used in addition to the IHA procedure in the characterization of the capsular status of the strains. Thirty-six strains or 90% were found to belong to type A and four or 10% to type D (Carter). The rapid agglutination (RA) test was employed to identify the-somatic antigens of the strains. Considerable cross reactions were experienced in conducting the RA test. Based upon the pattern of the cross reactions, at least 3 subgroups, viz., a, b, and c, were assigned to 0 group 1 of Namioka and Murata. Thirty strains were found to possess somatic antigen belonging to group 1. 0f the ten strains that were not satisfactorily identified, four were type D, four were auto- agglutinable (type A), and two were type A. These general findings. have important implications for the preparation of Pasteurella bacterins. Some of the cross reactions obtained with several antisera were thought to be due to a low level of "normal antibody" which was increased when the rabbits were repeatedly injected with killed Pasteurella organ- isms. To test this hypothesis, at the conclusion of the study 12 rabbits were examined and it was found that five of them possessed low to moder- ate titers to P. multocida with 0 group 1a,b,c. 50 51 To avoid cross reactions attributable to "normal antibody" in serological studies involving P. multocida, examination of sera prior to the inoculation of Pasteurella antigens is advisable. Sixty serum samples obtained from cattle prior to vaccination were examined for their PasteureZZa antibody titers.' Four samples were found to possess low titers of antibody to P. multocida type A. Seven samples possessed low to moderate amounts of antibody to 0 group 1a,b. After vaccination, there was no significant difference in the antibody, titers between control and vaccinated animals. Thus there was no evidence to indicate that a single dose of the Pasteurella bacterin in question produced a significant humoral immune response. 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(English Abstract), 13, (1934): 181-191. In Vet. Bull., 5, (1935): 114. APPENDIX APPENDIX Table A-1. Pasteurella antibody titers (RA test) in unimmunized rabbits Antigen. B-1476-7l M-14 (0 group. (0 group P-180-71 Serum No. 1a,b,c) 1a,b,c) (0 group 1a,b) l 20 20 20 2 40 160 80 3 -_ -_ -- 4 -_ __ -_ 5 _- __ _- 6 __ __ _- 7 -_ __ -_ 8 __ __ __ 9 4O 80 80 10 40 80 40 ll -- -- -- 12 10 20 -- Table A-2. Pasteurella antibody titers (RA test) in unabsorbed and absorbed antisera Serum M-6-l absorbed with Antigen M-6-l B-702 B- 702 320 -- M-5 « 320 20 M-6 320 -- B-1112-7l 320 -- P- 48-71 320 -- 58 HICHIGQN STQTE UNIV. LIBRARIES 31293 |||||||||l| III III“! Iii! Ill! ll Ill 01 0091043