Bordetella pertussis serotypes in Canada S. TOMA, MD, FRcP[c]; H. Lo, FIML5; M. MAGUS, BSA, DIP BACT A study was done to determine the major antigenic factors of Bordetella pertussis strains isolated throughout Canada and whether these isolates have the same antigenic structure as the bacilli in the currently used vaccines. Testing for the major pertussis antigens, factors 1, 2 and 3, was conducted with 440 freshly isolated strains of B. pertussis received from seven Canadian provinces between August 1976 and February 1978 and six batches of pertussis vaccine or immunizing agents containing pertussis vaccine. With the aid of specific antisera prepared in rabbits, the antigenic factors were detected by a slide agglutination technique. Almost all (98.90/o) of the pertussis strains examined were serotype 1,3. All six batches of pertussis vaccine or immunizing agents containing pertussis vaccine proved to be rich In each of the three main pertussis agglutinogens.

More than 50 years have passed since the first report of the successful use of pertussis vaccine to control an epidemic.1 Experience has shown that the use of pertussis vaccine in well organized mass immunization programs can reduce the incidence of whooping cough in children by as much as 50-fold, though doubt has periodically been cast on the effectiveness of certain batches of pertussis vaccine.2 The serotyping system for Bordetella pertussis based on the detection of agglutinogens has been in use since the identification of six pertussis antigens by Andersen3 and by Eldering, Hornbeck and Baker.4 The existence of three major pertussis antigens, factors 1, 2 and 3, and three major pertussis serotypes (1,2; 1,2,3; and 1,3) is now widely recognized.54 A change in the relative frequency of B. pertussis serotypes took place in Great Britain between 1953 and Une etude a 6t6 r6alisee dans le but 1966, the predominant serotype de d6terminer les facteurs antig6niques changing from 1,2 to 1,3. The same majeurs des souches de Bordetella pattern occurred in Australia, Canpertussis lsoi6es au Canada et aussi ada, the United States and most de verifier si ces isolats possedent countries in Europe. East Berlin is les mames structures antig6niques que one area in which the predominant celles des bacilies retrouv6 dans les serotype has remained 1,2,3 since vaccins d'utilisation courante. 1962.' La recherche des antig.nes majeurs de B. pertussis, les facteurs 1, 2 et 3, Interest in pertussis vaccines was a .t6 effectu6e sur 440 souches renewed after Preston7 reported that fraichement isoi6es et reques de sept the protection they conferred was provinces canadlennes entre ao0t 1976 related to each vaccine s content of et f.vrler 1978, et sur six lots de the prevalent serotypes. Most of the vaccin anticoquelucheux ou d'agent pertussis vaccines used in the United d'lmmunisatlon contenant un vaccin Kingdom in the 1950s and 1960s anticoquelucheux. A l'aide d'antis6rums contained antigenic factors 1 and 2 sp6cifiques pr6par6s chez le lapin, only. Because of the lack of factor les facteurs antlg.niques ont 6t6 ddtect6s par Ia technique d'aggiutlnation 3 (the prevalent antigenic factor at sur lamelle. that time in the United Kingdom) Presque toutes les souches de these vaccines conferred poor imB. pertussls examln6es (98.90/o) 6talent munity against pertussis.' du s6rotype 1,3. Tous les six lots de Pertussis vaccine was introduced vaccin anticoquetucheux ou d'agent Canada around 1936; immunizing in d'immunlsatlon contenant un vaccin agents containing diphtheria and peranticoquelucheux ont d6montr6 une forte tussis (DP), diphtheria, pertussis and teneur des trols agglutinog&nes majeurs tetanus (DPT), and diphtheria, perde B. pertussis. tussis, tetanus and polio were introduced in 1943, 1948 and 1959 reFrom the clinical bacteriology and special reference laboratories, provincial public spectively.'0 The first report on the health laboratories, Ontario Ministry of presence of antigenic factors 1, 2 and Health, Toronto 3 in strains of B. pertussis and in Reprint requests to: Dr. S. Toma, pertussis vaccines in Canada was Medical bacteriologist, Provincial public published in the Journal in 1965.11 Of health laboratories, P0 Box 9000, Terminal "A", Toronto, Ont. M5W iRS 58 strains examined (54 from the 722 CMA JOURNAL/OCTOBER 7, 1978/VOL. 119

Hospital for Sick Children and 4 from the Ontario provincial public health laboratories in Toronto) 56 were found by culture to be serotype 1,3, one was found to be 1,2 and one was found to be 1,2,3. All 10 pertussis vaccines examined at that time contained factor 1, 2 (possibly 4) contained no factor 2 and 7 contained little or no factor 3. Eleven strains of B. pertussis cultured in 1967 at the Windsor, Ont. regional public health laboratory were reported as serotype 1,3." Preston,'3 on the subject of the serotyping of other pertussis strains in Canada, said that Dr. A.J. Wort in Nova Scotia in 1971-72 isolated more than 60 strains of B. pertussis of serotype 1,3. In the same year in Toronto Cameron14 examined 12 fresh isolates of B. pertussis and found that the predominant serotype was 1,2,3 (in 9 isolates); subsequently, however, these isolates were re-examined (personal communication, 1978) and shown to be serotype 1,3. Fifty-five strains of B. pertussis were later isolated in the Ottawa area (in 1975-76) and found to be serotype 1,3.15 There appeared to be a need to monitor which B. pertussis serotypes are being isolated in Canada and to determine whether the currently used pertussis vaccines contain the antigenic factors of these serotypes. Material and methods Preparation of typing antisera Standard cultures: One set of lyophilized cultures of B. pertussis strains 3747 variant (serotype 7,1), 3865 (7,1,2,4), D35726 (7,1,3,6), B16 (7,1,3) and D41633 (7,1,2,*3,4) was obtained from the national collection of type cultures, Colindale, London, England; another set of cultures of B. pertussis strains 5373 (7,1,3,6,13), 5374 (7,1,2,5,6,13) and 5375 (7,1,2,4,13) and of B. bronchi-

septica variant 5376 (7,8,9,12,13) was received from the state public health laboratories, Grand Rapids, Michigan. The strains were cultured on charcoal agar medium (CM 119, Oxoid) and incubated for 48 hours *A weak agglutinogen.

Table I-Strains of BoTdetella pertussis and antisera used in study of current serotypes Antigenic factor Immunizing Absorption Agglutinins In monospecific strain strain removed antiserum 3747 variant 5376 7 1 3865 D41633 7,1,4 2 D35726 5374 7,1,6 3 5375 5374 7,1,2,13 4 5374 3865 7,2 5 5373 1,6,13 D35726 B16 7,1,3 6

Table Il-B. pertussis serotypes in Canada Source of cultures No. of cultures 1,3 Ontario 356 353 Toronto 275 Ottawa 50 Other 31 Alberta Edmonton 31 31 Manitoba WinnIpeg 22 20 Quebec 12 12 Montreal 11 Other 1 Saskatchewan 11 11 Regina 1 Saskatoon 10 New Brunswick Three localities 5 5 Nova Scotia Halifax 3 3 Total 440 435 *One culture gave a weak reaction with factor 2 antiserum.

Serotype 1,2 1

1,2,3 2


2* -








fresh isolates we lyophilized 11 pertussis cultures of serotype 1,3 strains between August 1976 and August 1977, stored them at room temperature and retyped them in January 1978. The cultures were plated out and 15 isolated colonies from each culture were subcultured to a pure growth. All 165 subcultures showed the same antigenic structure as the original strains. The six lots of vaccine from Connaught Laboratories yielded 4 + slide agglutination with each of the three major pertussis factors in dilutions of 1:1, 1:2 and 1:4. Further dilution of the vaccines was not suitable for the slide agglutination technique. Discussion Whooping cough continues to produce substantial illness in Canada despite the widespread use of pertussis vaccine, and there is much controversy about the true incidence of the disease and the efficacy of the current vaccines. Working in a public health bacteriology laboratory we are involved in two important aspects of pertussis; ensuring the bacteriologic diaguosis and determining the relative frequency of the organism's serotypes. Isolation of B. pertussis has long been a problem for our laboratory (which receives most specimens by mail or courier) and the problem was not resolved until August 1974. Until then most such isolations in the Toronto area had been made at the Hospital for Sick Children. Improvements in our isolation media and methods of collection and transport led to the reporting of 1419 isolations of B. pertussis and parapertussis by April 197620 and 2233 by the end of 1977. These isolations undoubtedly increased the reported figures for cases of whooping cough in Ontario and clearly indicate that unless the clinical diagnosis of whooping cough is backed by an efficient bacteriologic diagnosis gross under-reporting of cases is inevitable. We then tried to determine whether the antigenic factors in the strains of B. pertussis being isolated corresponded to those in the currently used vaccines. In Canada neither commercial typing antisera nor reference serotyping services were available. It was therefore necessary for us to prepare our own absorbed sera containing the major antigenic

STANDFAST AFB: The serotypes of factors 1, 2 and 3, which yielded speBordetella pertussis isolated in Great cific reactions with all control culBritain between 1941 and 1968 and a tures and were used in the serotyping comparison with the serotypes obstudy. Use of absorbed sera containserved in other countries during this ing the minor factors 4, 5 and 6 was period. J Hyg (Camb) 76: 265, 1976 discontinued when they were found 6. AGARWAL KC, PRESTON NW: Pertussis agglutinins in vaccinated mice: to produce some cross-reactions. difficulty in estimating the type-speThe finding that 98.9% of the cific response. indian J Med Res 64: 440 strains of B. pertussis tested 393, 1976 were serotype 1,3 is in agreement 7. PRESTON NW: Effectiveness of pertussis vaccines. Br Med J 2: 11, 1965 with serotyping data obtained in Idem: Technical problems in the laboCanada over the past 14 years.'1'3"' 8. ratory diagnosis and prevention of Accumulated evidence shows that, whooping-cough. Lab Pract 19: 482, following subculture or storage in the 1970 laboratory, some strains of B. per- 9. Public Health Laboratory Service Whooping-Cough Committee and tussis may undergo spontaneous pheWorking Party: Efficacy of whoopingnotypic or genotypic changes involcough vaccines used in the United ving loss of antigens.21 An important Kingdom before 1968. A preliminary problem, therefore, in producing an report to the director of the public health laboratory service. Br Med J effective pertussis vaccine is the 4: 329, 1969 control and detection of all three 10. WILSON RJ: Canada's experience with major agglutinogens in the final multiple antigens. Can J Public Health product. A test for these agglutino53: 457, 1962 gens would constitute a simple but 11. CHALVARDJIAN N: The content of 1, 2 and 3 in strains of Bordetella peressential precaution against loss of tussis and in vaccines. Can Med Assoc important antigenic factors from 92: 1114, 1965 otherwise suitable vaccine strains.'8 12. JELDERING G, HOLWERDA J, DAVIS A, However, the quantitative detection Ct al: Bordetella pertussis serotypes in of the three agglutinogens in a vacthe United States. Adv Appl Microbiol 18: 618, 1969 cine through the production of antibodies in laboratory animals is dif- 13. PRESTON NW: Prevalent serotypes of Bordetella pertussis in non-vaccinated ficult. Preston8 used a semiquantitacommunities. J Hyg (Camb) 77: 85, tive slide agglutination technique to 1976 show that the final vaccine is rapidly 14. CAMERON J: Problems associated with the control testing of pertussis vacand specifically agglutinated by each cine, in Advances in Applied Microof the three monospecific typing sera. biology, vol 20, PERLMAN D (ed), Except for a report by Chalvardjian Acad Pr, New York, 1976, p 62 in 1965,11 there are no published 15. ROSSIER E, CRAN F: Bordetella perCanadian data on the agglutinogens tussis in the National Capital Region: prevalent serotype and immunization in pertussis vaccines. Chalvardjian status of patients. Can Med Assoc J stated that "seven of ten vaccines 117: 1169, 1977 were weak or deficient in antigen 3." 16. HURRELL WK: Procedural Manual for It was indeed encouraging that the Production of Bacterial, Fungal and six batches of pertussis vaccine or Parasitic Reagents, rev 3rd ed, Center for Disease Control, Atlanta, Ga, immunizing agents containing pertus1976 sis vaccine manufactured by Con- 17. BRONNE-SHANBURY CJ: The importnaught Laboratories and tested by us ance of agglutinin production in mice proved to be rich in each of the three in the determination of the definitive major pertussis agglutinogens. serotype of Bordetella pertussis. J Hyg

References 1. MADSEN ST: Whooping cough: its bacteriology, diagnosis, prevention and treatment. Boston Med Surg J 192: 50, 1925 2. Immunization against whooping cough (E). WHO Chron 29: 365, 1975 3. ANDERSEN EK: Serological studies on H. pertussis, H. parapertussis and H. bronchisepticus. A cta Pathol Microbid Scand [A] 33: 202, 1953 4. ELDERING G, HORNBECK D, BAKER J:

Serological study of Bordetella pertussis and related species. J Bacteriol 74: 133, 1957 5. BRONNE-SHANBURY

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(Camb) 76: 257, 1976 18. Recommended Specifications for Microbiological Reagents, 3rd ed, laboratory division, national communicable disease center, Center for Disease Control, Atlanta, Ga, 1968, p B1-4 19. Recommended Methods for Evaluation of Microbiological Reagents, 2nd ed, laboratory division, national communicable disease center, Center for Disease Control, Atlanta, Ga, 1969, p D5-1 20. REGAN J, LOWE F: Enrichment medium for the isolation of Bordetella.

J Clin Microbiol 6: 303, 1977 21. STANBRIDGE TN, PRESTON NW: Varia-

don of serotype in strains of Bordetella pertussis. J Hyg (Camb) 73: 305, 1974

Bordetella pertussis serotypes in Canada.

Bordetella pertussis serotypes in Canada S. TOMA, MD, FRcP[c]; H. Lo, FIML5; M. MAGUS, BSA, DIP BACT A study was done to determine the major antigenic...
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