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Third Inter-American Congress ON

Brucellosis Held under the joint auspices of: The Inter-American Committee on Brucellosis The United States Committee on Brucellosis of the National Research Council The Pan American Sanitary Bureau Regional Office, World Health Organization

WASHINGTON, D. C. November 6-10, 1950

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Third Inter-American Congress ON

Brucellosis Held under the joint auspices of: The Inter-American Committee on Brucellosis The United States Committee on Brucellosis of the National Research Council The Pan American Sanitary Bureau Regional Office, World Health Organizatio

WASHINGTON, D. C.

November 6-10, 1950 j "`

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Prepared for publication by the Editorial Section -Pan American Sanitary Bureau Regional Office of the World Health Organization 1501 New Hampshire Avenue, N. W. Washington, D. C., U. S. A.

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS Held in Washington, D. C., Nov. 6-10, 1950

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Dr. Alice C. Evans, President, Inter-American Committee on Brucellosis, addressing the Congress.

Dr. Weslev W. Spink, President of the Third Inter-American Congress on Brucellosis and of the United States Committee on Brucellosis.

9

Group of Delegates. on their visit to the United States Animal Industry Laboratories at Beltsville, Md. accompanied by members of the laboratory.

FOREWORD The Third Inter-American Congress on Brucellosis represented a continuation of the effort to bring about a better understanding of animal and human brucellosis in the Western Hemisphere. The First Congress was held in 1946 in Mexico City, and the Second in 1948 in Argentina. When workers in the field of brucellosis gathered in Washington, D. C. in November 1950 for the Third Inter-American Congress on Brucellosis, a worldwide interest in the problems of brucellosis was expressed. Through the World Health Organization and the Food and Agriculture Organization, which are agencies of the United Nations, official representatives were present from Great Britain, several European countries and from Japan. Although the clouds of war hovered on the horizon, and bitter fighting was engaging armies in Korea, these scientists met in Washington to discuss the problems of brucellosis. Misunderstanding between individuals and nations often stems from ignorance of each other's problems. Though there remain many problems on brucellosis to be solved, the Third Inter-American Congress permitted a review of the disease as it exists in different countries, and a recital of advances that had been made in various clinics and laboratories. As the scientific sessions proceeded, it became quite apparent that the problems of brucellosis varies from country to country. A failure to appreciate these variations had accounted for much of the misunderstanding and disagreement that had existed prior to the Congress. As a result of the Congress, a more comprehensive picture of brucellosis as it exists throughout the world was crystallized. Equally important was the opportunity scientists had to meet in person and create friendships that are so essential in international relationships today. The following papers, with a few exceptions, constitute the program of the Third Inter-American Congress on Brucellosis. There has been no editorial revision of these papers; they are printed as submitted by the authors, and in order to publish the manuscripts at a minimum of expense and without too much delay, the authors have had no opportunity to proofread their papers. No discussions are included and all illustrations have been omitted. The members of the Congress are sincerely appreciative of the Pan American Sanitary Bureau's willingness to publish, at cost, this volume containing a compilation of the papers presented. WESLEY W. SPINr, M.D. President, Third Inter-American Congress on Brucellosis

ACKNOWLEDGMENTS

The scientific meetings of the Third Inter-American Congress on Brucellosis were made possible through the generous financial support of the following organizations in the United States: Abbott Laboratories Merck and Co., Inc. The Borden Company National Dairy Products Corp. Burroughs Wellcome and Co. Parke, Davis and Co. Pet Milk Co. Lederle Laboratories Division, American Cyanamid Co. Eli Lilly and Co. Chas. Pfizer and Co., Inc. E. R. Squibb & Sons The Pan American Sanitary Bureau provided the generous assistance of its staff, especially the services of the Veterinary Public Health Section Secretary General's Office Conference Section Public Health Division Office Services Section The National Research Council contributed to the success of the Congress, especially Dr. LeRoy Voris, Executive Secretary of the Agriculture Board; and the United States Department of Commerce by making available the Departmental Auditorium for the meetings.

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TABLE OF CONTENTS Page

Foreword . ............................................................... Acknowledgments......................... Alphabetical list of authors ............................................... Committees: Inter-American Committee on Brucellosis ................................ United States Committee on Brucellosis ................................. Pan American Sanitary Bureau ......................................... Alphabetical list of participants ........................................... Addresses: Address of Welcome Dr. Alice C. Evans .................................................... Welcome address Dr. Miguel E. Bustamante ............................................. Opening remarks Wesley W. Spink, M.D ................................................. Papers presented: Brucellosis-a World Problem Martin M. Kaplan, V.M.D., M.P.H.................................... Epidemiología de la Brucelosis Humana en la República Argentina; la Brucelosis del Este y del Oeste E. A. Molinelli, D. Ithurralde,G. Basso, S. Miyara, y C. P. Pandolfo...... Brucellosis Incidence in the United States James H. Steele and L. Otis Emik ...................................... La Brucelosis Humana en Cuba. Resumen del Estado Actual Arturo Curbelo y Viola Márquez ........................................ Estado Actual de la Brucelosis Bovina en Cuba Ana J. Pelaiz ......................................................... Animal Brucellosis in Brazil Milton Thiago de Mello ................................................ La Brucelosis Animal en el Uruguay B. Szyfres, J. L. Stella, W. Erradonea, H. Trenchi, D. Abaracón, J. C. Piñón, y J. M. Infantozzi .......................................... Brucellosis, A Packing Plant Problem Norman B. McCullough................................................ Variations as a Tool in Brucellosis Research W. Braun, A. Gorelick, M. Kraft and D. Mead ........................... Bactericidal Action of Human Blood Against Brucella and Its Specific Inhibition Wendell H. Hall ....................................................... The Clinical Course of Human Brucellosis in Minnesota Wesley W. Spink and Robert L. Magoifin ................................ The Natural Course of Bovine Brucellosis David T. Berman ...................................................... vii

v vi ix xiii xiii xiii xiii

1 3 5

6

15 28 37 48 59

69 76 83

87 94 104

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS Page

The Natural Course of Swine Brucellosis L. M. Hutchings....................................................... Studies of the Physical Properties and Agglutinability of Brucella Antigens Used in the Americas Fred Murdock, Martin H. Roepke and B.D. Blood ........................ *Field Studies on the Diagnosis of Animal Brucellosis with Special Emphasis on the Ring Test F. C. Driver and M. H. Roepke ........................................ Comparative Study by Media Ordinarily Used for the Growth of Brucella Species Genesio Pacheco and Milton Thiago de Mello ............................ The Carbon Dioxide Requirements of Brucella Abortus A. G. Marr and J. B. Wilson .......................................... Use of the Embryonating Egg for Isolation of Brucella in a Public Health Diagnostic Laboratory S. R. Damon and Kathleen Gay ........................................ Diagnóstico Bacteriológico y Biológico de la Brucelosis Luis A. Philipps...................................................... What Should Be Done with the Brucella Skin Test Karl F. Meyer ......................................................... Modalidades Clínicas de la Brucelosis Humana T. de Villafañe Lastra ................................................ Preparation and Properties of Brucella Abortus Vaccine (Strain 19) Desiccated by Lyophilization W. F. Verwey, N. H. Harringtonand Clara Matt ........................ Brucelosis Caprina en la República Argentina R. A. Maubecin, B. L. Morán, F. R. Jurado y V. C. Cedro ................ Controlling Brucellosis in Colorado Goats George W. Stiles....................................................... El Programa de Erradicación de Brucelosis en Puerto Rico Enrique R. Toro, Jr................................................... Control of Brucellosis in the United States B. T. Simms ......................................................... Trend of Nation-wide Brucellosis Eradication in the United States W. D. Knox ........................................................... Chemotherapy of Experimental Brucella Abortus Infection in Guinea Pigs and Mice A. Pomales-Lebrón, Howard E. Hall and C. Fernández.................. Estudios sobre Terapéutica de la Brucelosis M. Ruiz Castañeda,G. Guerrero Ibarray C. Carrillo Cárdenas ............ La Antibrucelina en el Tratamiento de la Brucelosis Febril Enso Criscuolo, F. Ramacciotti, Esther R. W. de Paolasso, H. Vacchiani, F. Bergagna, N. PierangeliVera y A. Ceballos ................. 285, Terramycin, Chloramphenicol and Aureomycin in Acute Brucellosis; A Preliminary Report John H. Killough, Gordon B. Magill and Richard C. Smith ................

115

122

133

145 151

157 165 177 191

209 216 226 238 244 252

260 276

288

296

ALPHABETICAL INDEX OF AUTHORS Page

Page

Driver, Fred C. U. S. Bureau of Animal Indus133 try, St. Paul, Minn ........... Emik, L. Otis 28 Scientist, Atlanta, Ga .......... Errandonea, W. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino", Dirección 69 de Ganadería, Uruguay........ Fernández, Carlos San Juan, Puerto Rico ........ 260 Fernández Ithurrat, E. M. Instituto Malbrán, Ministerio de Salud Pública, Buenos Aires, 15 Argentina..................... Gay, Kathleen Bureau of Laboratories,Indiana State Board of Health, Indian157 apolis, Ind .................... Gorelick, Arthur N. Camp Detrick, Frederick, Md... 83 Guerrero Ibarra, G. Departamentode Investigaciones Médicas, Hospital General, Mé276 xico, D. F., México ........... Hall, Howard E. 260 Baltimore, Md ................. Hall, Wendell H. Laboratory Service, Veterans Administration Hospitaland the Department of Medicine, University of Minnesota Medical 87 School, Minneapolis, Minn..... Harrington, N. H. Sharp and Dohme, Inc., Glenol-

Abaracón, D. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino", Dirección 69 de Ganadería, Uruguay........ Basso, G. Instituto Malbrán, Ministerio de Salud Pública, Buenos Aires, F 15 Argentina ..................... Bergagna, Fabio H. Clínica de Enfermedades Infecciosas, Facultadde Ciencias Médicas, Universidad de Córdoba, 288 Argentina..................... Berman, David T. Wisconsin Agricultural Experiment Station, University of Wisconsin, Madison, Wis .......... 104 Blood, Benjamin D. Pan American Sanitary Bureau Washington, D. C .............. 122 Braun, Werner Camp Detrick, Frederick, Md.. . 83 Carrillo Cárdenas, C. Departamento de Investigaciones Médicas, Hospital General, Mé- al 276 xico, D. F., México ............ Ceballos, Alberto Clínica de Enfermedades Infecciosas, Facultadde Ciencias Médicas, Universidad de Córdoba, 288 Argentina..................... Cedro, V. C. Ministerio de Agricultura y Ganadería, Buenos Aires, Argen216 tina ......................... Curbelo, Arturo Sección Experimental, Cátedra de Bacteriología, Universidad de 37 La Habana, Cuba ............. Criscuolo, Enso Clínica de Enfermedades Infecciosas, Facultad de Ciencias Médicas, Universidad de Cór285, 288 doba, Argentina ........... Damon, S. R. Bureau of Laboratories,Indiana State Board of Health, In157 dianapolis, Ind................

den, Penn.....................

209

Hutchings, Leslie M. Agricultural Experiment Station, Purdue University, Lafa115 yette, Ind ...................... Infantozzi, J. M. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino", Dirección 69 de Ganadería, Uruguay........ Ithurralde, D. Instituto Malbrán, Ministeriode Salud Pública, Buenos Aires, 15 Argentina..................... ix

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS Page

Jurado, F. R. Ministerio de Agricultura y Ganaderia, Buenos Aires, Argentina ......................... 216 Kaplan, Martin M. Division of Epidemiology, World Health Organization, Geneva, Switzerland ................... 6 Killough, John H. U. S. Naval Medical Research Unit No. 3, Cairo, Egypt...... 296 Knox, W. D. Editor, Hoard's Dairyman, Fort Atkinson, Wisc................ 252 Kraft, Mary Camp Detrick, Frederick, Md.. . 83 Magill, Gordon B. U. S. Naval Medical Research Unit No. 3, Cairo, Egypt ...... 296 Magoffin, Robert L. Department of Medicine, University of Minnesota Hospitals and Medical School, Minneapolis ........................... 94 Márquez, Viola Sección Experimental, Cátedra de Bacteriología, Universidad de La Habana, Cuba ............. 37 Marr, A. G. Departmentof Agricultural Bacteriology, University of Wisconsin, Madison, Wisc ............ 151 Matt, Clara Sharp and Dohme, Inc., Glenolden, Penn................... 209 Maubecin, R. A. Ministerio de Agricultura y Ganadería, Buenos Aires, Argentina ...................... 216 Mead, Dorothy D. Camp Detrick, Frederick, Md.. . 83 Mello, Milton Thiago de Army Veterinary Service, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil............. 59, 145 Meyer, K. F. George Williams Hooper Foundation, University of California, San Francisco, Calif........... 177 Miyara, S. Instituto Malbrán, Ministerio de Salud Publica,Buenos Aires, Argentina ..................... 15

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Molinelli, Ernesto A. Instituto Malbrán, Ministeriode Salud Pública, Buenos Aires, Argentina..................... 15 Morán, Benjamín L. Ministerio de Agricultura y Ganadería, Buenos Aires, Argentina ......................... 216 Murdock, Fred M. U. S. Bureau of Animal Industry, St. Paul, Minn........... 122 McCullough, Norman B. Microbiological Institute, National Institutes of Health, Public Health Service, Bethesda, Md ........................... 76 Paolasso, Esther R. W. de Clínica de Enfermedades Infecciosas, Facultad de Ciencias Médicas, Universidad de Córdoba, Argentina ............... 285 Pacheco, Genesio Instituto Oswaldo Cruz, Rio de Janeiro, Brasil................ 145 Pandolfo, G. Instituto Malbrán, Ministerio de Salud Pública, Buenos Aires, Argentina..................... 15 Pelaiz, Ana J. Centro de Brucelosis, Instituto Nacional de Higiene, Habana, Cuba ......................... 48 Pierangeli Vera, Néstor Clínica de Enfermedades Infecciosas, Facultad de Ciencias Médicas, Universidad de Córdoba, Argentina ............... 288 Piñón, J. C. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino", Dirección 69 de Ganadería, Uruguay........ Pomales Lebrón, A. San Juan, Puerto Rico ........ 260 Philipps, Luis A. Instituto Nacional de Biología Animal, Ministerio de Agricultura, Lima, Perú.............. 165 Ramacciotti, Félix Clínica de Enfermedades Infecciosas, Facultad de Ciencias Médicas, Universidad de Córdoba, Argentina ............... 285

ALPHABETICAL INDEX OF AUTHORS

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Page

Page

Roepke, Martin H. University of Minnesota, St. Paul, Minn............... 123, 133 Ruiz Castafeda, M. Departamento de Investigaciones Médicas, Hospital General, México, D. F., México ......... 276 Simms, Bennett T. Bureau of Animal Industry, Agricultural Research Administration, United States Department of Agriculture, Washington, D. C ...................... 244 Smith, Richard C. U. S. Naval Medical Research Unit No. 3, Cairo, Egypt ..... 296 Speroni, A. Instituto Malbrán, Ministerio de Salud Pública, Buenos Aires, Argentina..................... 15 Spink, Wesley W. Department of Medicine, University of Minnesota Hospitals and Medical School, Minneapolis ........................... 94 Steele, James H. Veterinary Director, Atlanta, Ga........................... 28 Stella, J. L. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino," Dirección de Ganadería, Uruguay........ 69

Stiles, George W. Laboratory Section, Colorado State Department of Public Health, Denver, Col ............ 226 Szyfres, B. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino," Dirección de Ganadería, Uruguay ........ 69 Toro, Enrique R., Jr. Negociado de Industria Animal, Bureau of Animal Industry, San Juan, Puerto Rico ....

238

Trenchi, H. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino," Dirección de Ganadería, Uruguay........ 69 Vacchiani, Hugo Clínica de Enfermedades Infecciosas, Facultad de Ciencias Médicas, Universidad de Córdoba, Argentina ............... 288 Villafañe Lastra, T. de Comisión Permanente para el Estudio de la Brucelosis, Circulo Médico de Córdoba, Córdoba, Argentina ............... 191 Verwey, W. F. Sharp and Dohme, Inc., Glenolden, Penn..................... 209 Wilson, J. B. Department of Agricultural Bacteriology, University of Wisconsin, Madison, Wisce ........... 151

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INTER-AMERICAN COMMITTEE ON BRUCELLOSIS President-Alice C. Evans, Washington, D. C. Secretary-M. Ruiz Castañfeda, Mexico City, Mexico UNITED STATES COMMITTEE ON BRUCELLOSIS (Committee on Brucellosis, National Research Council) W. W. Spink, University of Minnesota, Chairman W. L. Boyd, University of Minnesota C. F. Jordan, Tarrant County Health Dept., Fort Worth, Texas L. M. Hutchings, Purdue University C. K. Mingle, United States Department of Agriculture C. L. Larson, United States Public Health Service A. C. Evans, Washington, D. C. PAN AMERICAN SANITARY BUREAU Regional Ofgice, World Health Organization Fred L. Soper, Director J. R. Murdock, Assistant Director M. E. Bustamante, Secretary General B. D. Blood, Veterinary Public Health Section H. L. Hayward, Conference Section ALPHABETICAL LIST OF PARTICIPANTS Dr. E. M. Aldrich Inspector in charge of Tuberculosis and Brucellosis Eradication, Boston, Mass. Miss Virginia Allen State Laboratory of Hygiene, Madison, Wisc. Dr. Ramón Atalaya Cultural Attaché, Colombian Embassy, Washington, D. C. Dr. Juan Carlos Bacigalupi Chief, Municipal Bacteriological and Vaccine Service, Montevideo, Uruguay Dr. Henry Bauer Director, Medical Laboratories, Minnesota State Department of Health, Minneapolis, Minn. Dr. Alfonso Bautista Bustos Caracas, Venezuela Dr. Walter R. Baynes Assistant State Veterinarian, North Carolina Department of Agriculture, Raleigh, N. C. *Dr. Hans C. Bendixen Royal Veterinary College, Bülowsvej 13, Copenhagen, Denmark * Member of WHO/FAO Expert Panel on Brucellosis. xiii

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS Dr. Ramón S. Berges Cultural Attaché, Dominican Republic Embassy, Washington, D. C. Dr. David T. Berman Assistant Professor Veterinary Science, University of Wisconsin, Madison, Wisc. Dr. Irving H. Borts Laboratory Director, State Hygienic Laboratory, Iowa State Department of Health, Iowa City, Ia. *Dr. Werner Braun Biological Department, Chemical Corps, Camp Detrick, Frederick, Md. Dr. C. E. Brooke Chief, Clinical Investigations Branch, Camp Detrick, Frederick, Md. Dr. Harold S. Bryan Assistant Professor of Veterinary Pathology and Hygiene, College of Veterinary Medicine, Urbana, 1ll. Dr. Bert J. Cady Veterinarian in charge, Bureau of Animal Industry, Augusta, Me. Dr. Hugh S. Cameron Professor of Veterinary Medicine, University of California, Davis, Calif. *Dr. Birdsall N. Carle Chief, Brucellosis Unit, Microbiological Institute, National Institutes of Health, Bethesda, Md. Dr. Charles M. Carpenter Professor of Infectious Diseases, School of Medicine, University of California, Los Angeles, Calif. Dr. Clemente Carrillo-Cárdenas Assistant Chief, Medical Research Laboratory, Hospital General, México D. F., Mézxico Dr. Alejandro Chediak Professor, School of Medicine, University of Habana, Habana, Cuba Dr. C. W. Christensen Associate Director of Laboratories, Difco Laboratories, Detroit, Mich Dr. Alfred Cohn Bacteriologist, Montefiori Hospital, New York, N. Y. Dr. Marion B. Coleman Senior Bacteriologist, Department of Laboratory and Research, New York State Health Department, Albany, N. Y. Dr. Elvin Coon Veterinarian in charge, U. S. Bureau of Animal Industry, Charleston, W. Va. Dr. Cornelia Cotton Research Bacteriologistin Brucellosis, Livestock Sanitary Service, University of Maryland, College Park, Md. Dr. Enso Criscuolo Professor of Infectious Diseases, School of Medicine, Córdoba, Argentina. *Sir Weldon Dalrymple-Champneys, Bt. Deputy Chief Medical Officer, Ministry of Health, Whitehall, London, U. K. Dr. K. S. R. Damon Director of Laboratories, State Board of Health, Indianapolis, Ind.

COMMITTEES AND PARTICIPANTS

xv

Dr. I. S. Danielson Assistant Director of Animal Industry, Lederle Laboratorios,Division of American Cynamid Co., PearlRiver, N. Y. *Dr. Rebbi Durusan Head, Brucellosis Laboratory, Institute of Veterinary Bacteriology, Etlik, Ankara, Turkey Dr. Sanford Elberg Associate Professor of Bacteriology, University of California, Berkeley, Calif. Dr. L. Otis Emik Scientist (R), Communicable Disease Center, U. S. Public Health Service, Atlanta, Ga. Dr. José Carlos Falçao de Melo Technical Assistant of the Army, Biology Institute, Sao Paulo, Brazil Dr. Alberto Eduardo Fors Habana, Cuba Dr. E. R. Gentry Chief, Medical Division, Department of Medicine and Surgery, Veterans Administration, Washington, D. C. Dr. Herbert L. Gilman Professor of Bacteriology, New York State Veterinary College, Cornell University, Ithaca, N. Y. Dr. Kenneth Goodner Professor of Bacteriology, Jefferson Medical College, Philadelphia, Penn. Dr. Arthur N. Gorelick Medical Bacteriologist, Camp Detrick, Frederick, Md. Dr. Paulo Gouvea Department of Health, Niteroi, Brazil. Dr. Charles G. Grey Office of Experiment Stations, U. S. Department of Agriculture, Washington, D. C. Dr. Joseph Franklin Griggs Managing Director, Claremont Medical Research Foundation, Claremont, Calif. Dr. Patrick D. L. Guilbride Veterinary Surgeon, Department of Agriculture, Hope Kingston, Jamaica, B. W. I. Dr. Harold T. B. Hall Veterinary Consultant of FAO, Washington, D. C. Dr. Wendell H. Hall Chief, Bacteriology Section, Veterans Administration Hospital, Minneapolis, Minn. *Dr. Harold J. Harris Reserve Consultant in InternalMedicine, U. S. Naval Hospital, St. Albans, N. Y. Dr. Raymond J. Helvig Veterinarian, U. S. Public Health Service, Washington, D. C. Dr. Stanley L. Hendricks Public Health Veterinarian, Iowa State Department of Health, Des Moines, la.

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS Dr. Luis Alfredo Herrera Rodriguez Chief, Sanitary Inspection Section, Ministry of Public Health, Caracas, Venezuela Dr. David A. Hill State Supervisor, Brucellosis Control, Department of Agriculture, Columbus, O. Dr. Nell Hirschberg Bacteriologist,North CarolinaState Laboratoriesof Hygiene, Raleigh, N. C. Dr. Gladys L. Hobby Brooklyn, N. Y. Dr. Monroe A. Holmes Senior Assistant Veterinarian, U. S. Public Health Service, State Department of Health, Seattle, Wash. Dr. I. Forest Huddleson Professor of Bacteriology and Public Health, Michigan State College, East Lansing, Mich. Dr. Charles A. Hunter, Director of Laboratories, Kansas State Board of Health, Topeka, Kan. *Dr. Marcel M. J. Janbon Infectious Disease Clinic, School of Medicine, Montpellier, France Dr. Herbert John Jenne Chief, Bureau of Brucellosis Control, New Jersey Department of Agriculture, Trenton, N. J. Dr. Edwin P. Johnson Animal Pathologist, Virginia Agricultural Experimental Station, Blackburg, Va. Dr. Mary Barnette Johnson Senior Bacteriologist, Bacteriology Division, Alabama Public Health Department, Montgomery, Ala. *Dr. Christian L. Jorgensen Aarhus, Denmark Dr. James C. Kakavas Professorof Bacteriology, University of Delaware, Newark, Del. Dr. Martin M. Kaplan Veterinary Officer, World Health Organization, Geneva, Switzerland Dr. Emily H. Kelly Bacteriologist, Camp Detrick, Frederick, Md. Dr. J. Kiser Lederle Laboratories, Pearl River, N. Y. Dr. William D. Knox Secretary, National Brucellosis Committee, Ft. Atkinson, Wisc. Lt. Col. Ludwig R. Kuhn MSC Chief, Department of Bacteriology, Army Medical Department Washington, D. C. Mrs. Edith Kuhns Director, BacteriologicalLaboratory, State Board of Health, Helena, Mont. Dr. A. K. Kuttler Veterinarian in charge, Brucellosis and T. B. EradicationDivision, U. S. Bureau of Animal Industry, Department of Agriculture, Washington, D. C.

COMMITTEES AND PARTICIPANTS

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Dr. Félix Luciani Lairet Chief, Biological Productions, Institute of Hygiene, Caracas, Venezuela Dr. Martín A. Landa Habana, Cuba Dr. J. D. Leaming Lederle Laboratories,Pearl River, N. Y. Dr. Frank M. Lee State Public Health Veterinarian, Columbia, S. C. Dr. Ralph W. LeGrow Professor of Research, Ontario Veterinary College, Guelph, Ont., Canada *Dr. Israel Live Associate Professor of Bacteriology, University of Pennsylvania, Philadelphia Dr. Owen L. Lockwood Inspector in charge of Brucellosis and Tuberculosis Eradication in the States of Maryland and Delaware, Baltimore, Md. Dr. Leo Lowbeer Director of Laboratories, Hillcrest Memorial Hospital, Tulsa, Okla. Dr. C. D. Lowe Bureau of Animal Industry, U. S. Department of Agriculture, Washington, D. C. Dr. Frank H. Manley Professor of Infectious Diseases, Alabama Polytechnic Institute, Auburn, Ala. Dr. Charles H. Mann Medical Director of Heyden Chemical Corp., New York, N. Y. Dr. C. Manthei U. S. Bureau of Animal Industry, Animal Disease Station, Beltsville, Maryland *Dr. Giuseppe Mazzetti Professor of Hygiene, Institute of Hygiene, University of Florence, Florence, Italy Dr. José González Medina Department of Agriculture, Caracas, Venezuela Dr. Marco Antonio Mena Brito Chief, Laboratories, Children's Hospital, Mexico, D. F. Dr. Karl F. Meyer Director, Hooper Foundation, San Francisco, Calif. Dr. C. A. Mitchell Dominion Animal Pathologist, Department of Agriculture, Animal Disease Research Institute, Hull, Quebec, Canada Dr. P. Morales-Otero San Juan, Puerto Rico *Dr. Benjamin Lucas Morán Chief, Division of Brucellosis and T. B., Ministry of Agriculture, Buenos Aires, Argentina Dr. Charles E. Morris Assistant Inspector in charge, Veterinary Livestock, Albany, N. Y.

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Dr. Fred M. Murdock In charge of Production and Research, Anchor Serum Co., South St. Joseph, Mo. *Dr. Norman B. McCullough Surgeon, U. S. Public Health Service, Chicago, Ill. Mrs. Nell J. McNulty Hospital Bacteriology Department, Veterans Administration, Little Rock, Ark. *Dr. Leo Olitzki Professor of Bacteriology, Hebrew University, Jerusalem, Israel Dr. Galo Oliva Director General of Livestock, Ministry of Economy, Quito, Ecuador Mrs. Grace O'Neill Executive Offcer, U. S. World Federationfor Mental Health, New York, N. Y. Dr. Carlos Ortiz Mariotte Chief, Department of Epidemiology, Ministry of Health and Welfare, Mexico D. F., Mexico *Dr. Genesio Pacheco Oswaldo Cruz Institute, Rio de Janeiro, Brazil Dr. Ana Peláiz de Arán Chief, Brucellosis Center, Ministry of Health, Habana, Cuba Comdr. Frank R. Philbrook, MC, USN Research and Development Board, Department of Defense, Washington, D. C. Dr. Américo Pomales-Lebrón Professor of Bacteriology, University of Puerto Rico, San Juan, Puerto Rico Dr. Oswaldo Portella Assistant of the National School of Medicine, Rio de Janeiro, Brazil Dr. Horace M. Powell Head of Bacteriology, Lilly Research Laboratories, Indianapolis,Ind. Lt. Col. William D. Preston Chief, Internal Medicine Department of the Air Force, Washington, D. C. Dr. Jorge Quirós Sáenz, (Representing the Government of Costa Rica), College Park, Md. *Dr. Gérard Renoux Microbiology Laboratory, School of Medicine, Montpellier, France Dr. Mila E. Rindge Epidemiologist, Connecticut State Department of Health, Hartford, Conn. Dr. Alfonso Rivera Chief Veterinarian, Department of Agriculture and Commerce, San Juan, Puerto Rico Comdr. Robert E. Rock Bureau of Medicine and Surgery, U. S. Navy, Washington, D. C. *Dr. Martin H. Roepke School of Veterinary Medicine, University Farm, St. Paul, Minn. *Dr. Maximiliano Ruiz-Castañeda Director, Institute of Medical Research, General Hospital, Mexico, D. F. Mexico Dr. Kogi Saito Chief, Animal Hygiene Section, Ministry of Agriculture and Forestry, Tokyo, Japan

COMMITTEES AND PARTICIPANTS

xix

Dr. John H. Scruggs Veterinary Officer, U. S. Public Health Service, Indiana State Board of Health, Indianapolis, Ind. Dr. Sidney Silverman Bacteriologist, Camp Detrick, Frederick, Md. *Dr. Bennett T. Simms Chief, Bureau of Animal Industry, Department of Agriculture, Washington, D. C. Dr. Lawrence W. Slanetz Professor of Bacteriology, University of New Hampshire, Durham, N. H. *Dr. Arthur Wallace Stableforth Director, Veterinary Laboratory, Ministry of Agriculture, New Haw, Weybridge, England *Dr. James H. Steele Chief, Veterinary Public Health Service, Communicable Disease Center, Atlanta, Ga. Dr. George W. Stiles Director, Laboratory Section, Colorado State Department of Public Health, Denver, Colo. Dr. Luis Francisco Thomen Ambassador of the Dominican Republic to the United States, Washington, D.C. *Dr. Axel Thomsen Chief, Department of Veterinary Medicine, State Veterinary Serum Laboratory, Copenhagen, Denmark Dr. W. T. S. Thorp Chief, Comparative Pathology and Hematology Section, National Institutes of Health, Bethesda, Md. Dr. Enrique R. Toro, Jr. Assistant Veterinarian in charge, U. S. Bureau of Animal Industry, San Juan, Puerto Rico Dr. W. F. Verwey Director of Bacteriology Research, Medical Research Division, Sharp and Dohme, Philadelphia, Penn. Dr. Kenneth F. Wells Associate Chief Veterinarian, Dominion Department of Agriculture, Ottawa, Ont., Canada Dr. Joe Bransford Wilson Associate Professor, Department of Agricultural Bacteriology, University of Wisconsin, Madison, Wisc. Dr. W. H. Wiswell Assistant to Veterinarian in charge, Brucellosis and T. B. Eradication, Baltimore, Md. Dr. Teresa T. Woo Pediatrician,Health Department, Washington, D. C. Dr. Samuel H. Work Experimental Station, Department of Agriculture, Washington, D. C. Dr. George G. Wright Chief, Immunology Brucellosis, Camp Detrick, Frederick, Md. Dr. Adalbert O. Zink Lederle Laboratories, New York, N. Y.

ADDRESS OF WELCOME By Dr. AICE C. EVANS President, Inter-American Committee on Brucellosis It is a high honor and a great pleasure for the President of the Inter-American Committee on Brucellosis to have this opportunity to welcome to her home city the Third Inter-American Congress on Brucellosis. The Congress was organized by the United States Committee on Brucellosis of the National Research Council, which acts also as our national branch of the Inter-American Committee. I shall speak for a few moments as a member of the Organizing Committee. You honor us by your presence here, and we thank you for coming. We want this to be a week of benefits to all in the sharing of knowledge and experience in brucellosis problems. And we want it to be a week when new ties of international friendship are formed, and old ties are strengthened. In addition to the scientific program, a social program has been arranged which will provide an opportunity for an informal exchange of ideas about brucellosis problems. At these social gatherings there will be opportunity to enhance the appreciation of cultural differences which always grace and enliven international meetings. We want you to enjoy our city. We hope that you will find the time to see something of our national shrines, our government buildings, our art galleries and our parks. One of the features of this city in which we scientists take especial pride is its technical libraries. You are welcome to use them during your visit here. The Library of the United States Department of Agriculture and the Army Medical Library, both located within a few blocks from this building, and also the Library of Congress may hold source material which for a long time you have wanted to find. The staffs of those libraries will be eager to help you in the search for any information which you may want. We have a Hospitality Committee with members who speak Spanish and Portuguese. This committee has a desk near the registration desk. We hope that you and the members of your family who accompany you will make full use of this service in obtaining any kind of information which you may want. Since many of you are attending an Inter-American Congress on Brucellosis for the first time, a brief résumé of its historical background may be appropriate. In May, 1939, the First National Congress for the study of Malta Fever was held in the city of Torreon, Mexico. Continuing the series, the Fifth Mexican Congress was held in Mexico City in October-November, 1946. Invitations were sent to many leading scientists of North and South America who were interested in brucellosis. Delegates attended from 8 American countries. The First Inter-American Congress on Brucellosis grew out of the Fifth Mexican Congress when an Inter-American Committee of three members was elected. A few days after the close of the First Congress Dr. Mazza, the Argentine member of the Inter-American Committee, passed away. Homage was paid to his memory and a plaque was placed on his tomb in exercises held during the 1

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Second Congress held in Buenos Aires in 1948. Your Committee felt keenly the loss of Dr. Mazza's counsel. His place should be filled by election at the final session of this Congress. The purposes of this Inter-American organization are to stimulate research into brucellosis problems and to facilitate the dissemination of information gained through research. To accomplish these purposes the Inter-American Committee was charged with two chief duties: the promotion of a series of congresses and the organization of. national committees on brucellosis in the various countries. Each of the national committees has the responsibility of stimulating the study of brucellosis problems and the dissemination of information on brucellosis in the respective country. The national committee is the medium through which the international organization reaches the scientists and other interested persons. National committees have been organized in 13 countries, as follows: Argentina, Bolivia, Brazil, Canada, Colombia, Cuba, Chile, United States, Mexico, Panama, Peru, Puerto Rico and Venezuela. (The listing is in the alphabetical order determined by the Spanish spelling, as adopted by the Pan American Sanitary Bureau.) It is hoped that those countries which have not yet organized a national committee will do so in order that they may take active part in the growing movement. The Second Inter-American Congress on Brucellosis was held in Argentina in 1948, with delegates present from eight American countries. Sessions were held in Mendoza, November 17 to 20, and in Buenos Aires, November 22-26. The Congress was held under the auspices of the Government of Argentina and the Pan American Sanitary Bureau, in collaboration with the Inter-American Committee. During the Second Congress plans were made for the present Congress, which is held under the joint auspices of the United States Committee on Brucellosis of the National Research Council, the Pan American Sanitary Bureau and the Inter-American Committee on Brucellosis. We are happy to welcome to this Third Congress the European delegates who represent the World Health Organization. From the beginning the Inter-American Committee has borne in mind that when the World Health Organization should develop, brucellosis problems would force themselves upon it. The intention of the Inter-American Committee was always to adjust itself to play a part in the world-wide organization. The relationship between the two organizations is a matter which should be under your consideration this week. The InterAmerican Committee will appoint a subcommittee to meet with representatives of the World Health Organization to work out a plan for the future status of the Inter-American Committee in relationship to WHO. The frequency of meeting is a subject which should be reconsidered. Some members of the U. S. Committee on Brucellosis think that the interval between meetings should be longer than two years. Discussion and action on these matters should take place at the final session of this Congress. In closing, may I say again that we heartily welcome you all into our midst.

WELCOME ADDRESS By Dr. MIGUEL E. BUSTAMANTE

Secretary General, Pan American Sanitary Bureau Dr. Evans, Delegates to the Third Inter-American Congress on Brucellosis, ladies and gentlemen: The Pan American Sanitary Bureau, Regional Office of the World Health Organization for the Americas, because of its continued interest in brucellosis and the important part it plays in its health program for the Americas, has sponsored and assisted in the preparation of this Third InterAmerican Congress on Brucellosis. It is gratifying to add the name of the capital of the United States to those of Argentina and Mexico where the two former Congresses on Brucellosis, the First and Second, were held in 1946 and 1948, respectively, and in the preparation of which the Bureau also assisted. The Director of the Pan American Sanitary Bureau, Dr. Fred L. Soper, who is absent, has requested me to convey to you his regrets at being unable to welcome you to this city of Washington where the headquarters of the Bureau are located. Here, the Veterinary Public Health Section of the Division of Public Health devotes much time and effort in an endeavor to solve the many problems related to the international aspects of zoonoses, mainly brucellosis, a disease which greatly concerns not only the Departments of Health and of Agriculture, but the medical and veterinary professions as well. International gatherings such as this afford excellent opportunities for the interchange of ideas and discussion of the scientific, social and economic implications of a disease which plays such havoc with human and animal lives alike. Scientists all over the world are enabled to benefit by such discussions, by the results obtained thereof, and from the papers presented by their colleagues; all of which serves to stimulate them to carry on their active work with added zest. Brucellosis is a menace to man and domestic animals, impairs health in many ways and its multiple and variable behavior is a source of concern to clinicians, veterinarians, microbiologists and pathologists. Answers to the ever increasing number of questions regarding both diagnosis and treatment are yet to be supplied by the laboratory and the hospital. The importance of public health to the development and welfare of society is evidenced in diseases like brucellosis which present an example of a pathological problem to man and animals. Aside from its seriousness as an individual disease, brucellosis hampers the advancement of nutritional standards and endangers likewise the development of animal industry and of agriculture. The principles of prevention, control and eventual eradication of disease in general apply to the measures taken to cope with the problem of brucellosis in goats, pigs and cattle, to the studies made to check its spread to other domestic or wild animals, and above all to prevent by every possible means the spread of this disease among men. Clinical and laboratory research studies are being undertaken throughout the world by you, the members of the medical, veterinary and allied professions, and progress demands the exchange of such knowledge through the media of dis3

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

cussions, explanatory talks or suggestions, as well as through the adoption of resolutions at congresses of this nature, in order that the results obtained therefrom may be brought to the consideration of the World Health Organization and of the Pan American Sanitary Bureau. It is fitting, therefor, to expect that this Congress shall motivate the results we are all seeking in terms of better health for each and every one of the countries throughout the Americas. In welcoming you to Washington, congratulating ourselves for having you in our midst, I have the pleasure of extending to you the greetings of the Director and of the professional staff of the Bureau, and also of inviting you to visit our headquarters in this city.

OPENING REMARKS By WESLEY W. SPINK, M.D.

Chairman, United States Committee on Brucellosis Dr. Evans, members of the Congress and guests: On behalf of the United States Committee on Brucellosis I am glad to welcome you to the Third InterAmerican Congress on Brucel]osis. Two years ago the Committee on Public Health Aspects of Brucellosis of the National Research Council was designated as the American Committee on Brucellosis and entrusted with the responsibiilty of organizing the scientific sessions of this Congress. The Committee has worked hard to present a representative program embracing the various aspects of the brucellosis problem. Many of you have come a long way to attend this meeting, and we hope that the effort will prove worthwhile. In organizing this Congress the Committee has had the aid of many individuals and groups, without whose assistance this meeting could not have been held. All the financial support for the Congress has come from private sources; in fact, a group of private industries in the United States provided the necessary money. With this fund we have been able to provide a simultaneous translation service such as is used at the meetings of the United Nations. Each paper and discussion will be presented in English and in Spanish. This fund has also provided for a reception to be held in the Pan American Union this evening. The Committee is desirous of publishing the papers presented at this Congress as soon as possible. Since there are not enough available funds for this purpose, a registration fee of $5.00 is being charged for each person who desires a copy. In order to keep down the expense of the publication no discussions will be included. The American Committee on Brucellosis is also deeply indebted to the personnel of the Pan American Sanitary Bureau, especially to Dr. Miguel E. Bustamante and to Dr. B. D. Blood. This Congress could not have been held without the aid of this group. In addition, we have received considerable assistance from the Division of Biology and Agriculture of the National Research Council, particularly Dr. LeRoy Voris, Executive Secretary of the Agricultural Board. Each paper will be limited to 20 minutes for presentation and each discusser will be allowed 5 minutes. Adherence to this time limit is essential if the program is to be completed within a reasonable length of time. Although differences of opinion will most certainly arise as the meeting progresses, let the discussion be frank but friendly.

5

BRUCELLOSIS-A WORLD PROBLEM By MARTIN M. KAPLAN, V.M.D., M.P.H. Division of Epidemiology, World Health Organization, Geneva, Switzerland The organization of three Inter-American Congresses on Brucellosis in the past several years attests to the increasing awareness of public health workers to the magnitude of brucellosis as an international problem. A cursory glance at the available statistics on this disease, particularly when they are compared with those of other communicable diseases like tuberculosis and malaria, might give the impression that we are unduly concerned about this problem. Unlike tuberculosis and malaria, however, which patently afflict large sections of the world's population, the ravages of brucellosis are in great part still not recognized as stemming from a specific entity, because of the insidious nature of the disease and the difficulties involved in its diagnosis. Thus, brucellosis might be compared to an iceberg, since only its peak is visible to the casual observer. However, even our limited knowledge of this infection reveals that the prolonged physical suffering and reduced work capacity caused by brucellosis affect large segments of agricultural workers and other exposed groups, and that huge economic losses are resulting from decreases in milk production and breeding efficiency in affected livestock. In France, for example, it is estimated that the economic losses from brucellosis infection based on work days missed, care of the sick, and decreases in meat and milk production, exceed 100 million dollars annually;' in the United States the same figure is estimated for annual losses to the agricultural economy alone, apart from the human element involved. 2 These facts urge on us the conclusion that, in restoring and improving the health and economic well-being of countries throughout the world, brucellosis must be given primary consideration. Realizing the importance of the problem, various member countries of the United Nations have requested the specialized agencies, WHO and FAO, concerned with disease problems in man and animals to co-ordinate an international attack on brucellosis. Let us first examine briefly the known prevalence of brucellosis at the present time. Since this Congress will hear other papers on the status of brucellosis in North and South America, and most of you are aware of the general scope of the problem in these countries, we shall be concerned here principally with areas outside the Western Hemisphere. Table I is the officially reported incidence of human brucellosis infection in 1949. 3 6

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BRUCELLOSIS-A WORLD PROBLEM

The number of undiagnosed and unreported cases was undoubtedly far greater than these official figures. Thus, despite the fact that only 4000 to 6000 cases per year for the past several years have been officially reported in the United States, it has been estimated that the number of cases of brucellosis occurring each year in this country is perhaps between 40,000 and 100,000.4 Comparable figures for France are 1400 reported, and a minimum of 9000 cases as having actually occurred last year.' When one considers the nature of this disease, which may last over a period of years, the implications of these figures become more apparent. TABLE I-Reported cases of brucellosis in man for 1949 Continent

Africa

America

Country

Algeria Eritrea French Morocco Tripolitania Canada Chile Costa Rica Cuba Mexico Peru Unites States

Asia

Indo-China Irak

Oceania

Australia Hawaii New Zealand

Cases

Continent

Country

46

Europe

Austria Belgium Denmark France Germany Greece Ireland Italy Malta Netherlands Norway Poland Spain Sweden Switzerland Trieste Turkey Yugoslavia

42

9 9 188 32* 3* 28 1369* 491 4124 6 1* 38 4 30

Cases

24 8 196 1400 207 75* 6* 9426

910* 14 0 32 5484 20 196 2* 26* 73

*Incomplete.

Information concerning the prevalence of brucellosis infection in animals is at the present time very incomplete. Published reports are usually based on local surveys of a very limited nature. For the purposes of this paper, therefore, it was decided to send personal inquiries to government officials and brucellosis authorities in various countries outside the Western Hemisphere. The outline given below summarizes the answers received to date, and also includes personal observations of the author. References are used where reports in literature have been consulted. AFRICA Belgian Congo.-Bovine and caprine brucellosis sporadic in different parts of colonies. Approximately 7% of pregnant cattle in more highly developed areas of colonies abort, probably due in large part to brucellosis. In the region of

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Stanleyville, Br. Abortus isolated from cow, and 20% of 200 tested animals were positive reactors. Egypt.-Br. abortus infection common in cows and buffaloes; extent unknown. French Somaliland.-Apparently no bovine brucellosis; suspected in sheep and goats, but never confirmed, and extent in these animals is unknown. Madagascar.-Apparently no indigenous infection; one case reported in cow recently imported from France. Union of South Africa.5 -Between 25 and 50% of bulk milk reaching urban communities shows positive ring tests. Very few brucellosis-free areas in the country. Br. abortus prime offender; Br. melitensis believed to be present although not confirmed as yet. Fairly numerous human infections have been reported from Eritrea, Algeria, Kenya, Morocco, Tunis and Tripolitania; extent of animal infection in these countries unknown. Animal infection known to exist in Rhodesia, Ethiopia and Mauritius. ASIA China.-Br. melitensis and Br. abortus known to exist in human beings and animals; extent unknown. India. 6-Disease found in 10 to 50% of village cattle in areas of heavy rainfall; Br. abortus infection usual, but approximately 40% of Indian field strains had typing characteristics intermediate between Br. abortus and Br. melitensis. Israel.-Only few herds are infected at present. Japan.-Br. abortus infection in cattle fairly common; extent unknown. Br. suis isolated in 1940. Siam.-No cases of brucellosis in man or animals reported. Turkey.7-Approximately 18% of bovine found infected out of survey comprising over 8000 animals. Both Br. abortus and Br. melitensis infection exist in cattle and buffalo. Br. melitensis infection common in sheep and goats. Animal infection known to exist in Irak, Israel, Lebanon, Ceylon, Pakistan, Indo-China, Indonesia and Philippines; extent in these countries unknown. EUROPE Austria.-Approximately 20% of cattle infected. Belgium.-Approximately 5 to 6% of cows abort each year primarily due, it is believed, to brucellosis. Bulgaria.-Br. melitensis infection very common in sheep and goats; Br. abortus infection troublesome in herds around urban centers; extent of infection in animals unknown. Cyprus.-Free of brucellosis. Czechoslovakia.-Between 30 and 40% of cattle herds infected. Denmark.-Approximately 11% of cattle herds infected. Eire.-Br. abortus infection common in cattle; extent unknown. Finland.8-As of January 1, 1949, 627 infected herds. France.-Fifteen to 20% of cattle herds infected with Br. abortus (one au-

BRUCELLOSIS-A WORLD PROBLEM

9

thority gives this figure as exceeding 33%9); 15 to 40% of sheep and goat herds infected with Br. melitensis. Germany.9-Twenty-five to 50% of cattle herds infected. Greece.-Br.melitensis infection very common in sheep and goats; Br. abortus infection troublesome in herds around urban centers; extent of infection in animals unknown. Hungary.-Br. abortus infection common in cattle; extent unknown. Italy.-Br. abortus infection common in cattle of northern plains and large milking herds in northern Italy; approximately 30 to 40% of these herds infected. Cattle in central and southern Italy frequently infected with Br. melitensis. Sheep and goats are heavily infected in the mountainous areas of northern Italy; the central and particularly the southern portions of the country are likewise infected. Malta.-Cattle and goats heavily infected; Br. melitensis predominant. Netherlands.-Thirty to 40% of herds infected; approximately 8% of total number of cattle infected. Norway.-Disease eradicated except for two infected herds of cattle. Poland.-Between 25 and 35% of cattle herds infected. Rumania.-Cattle brucellosis known to be present; extent unknown. Spain.-Br. melitensis infection predominant. Cattle, sheep and goats heavily infected; extent unknown. Sweden.-Approximately 1% of cattle infected out of a total of 370,000 animals. Switzerland.9-Twenty-five to 40% of cattle herds infected. United Kingdom.-No recent data on prevalence of infection. In 1939 a minimum of 20% of the cattle in England were infected. Present policy of vaccination is believed to have reduced this figure considerably. Br. abortus only variety found, apart from a few isolations of certain strains which are indistinguishable, biochemically and serologically, from Br. melitensis. USSR.-Brucellosis in cattle, pigs, sheep and goats known to be troublesome in European and southern part of USSR; extent unknown. Yugoslavia.-Br. melitensis infection in cattle, sheep and goats very heavy in north-western part of country; disease believed widespread throughout remainder of country but extent unknown. Approximately 5% of cattle herds believed infected with Br. abortus. OCEANIA Australia.-Br. abortus infection in cattle fairly common; extent unknown. Hawaii.-Human infections reported but extent of disease in animals unknown. New Zealand.'O-Average annual incidence of abortions in dairy cattle of all ages is approximately 5%, apparently attributable in large part to brucellosis infections. The incompleteness of the above information illustrates the woeful lack of knowledge on the prevalence of brucellosis in large areas of the world. It is hoped that some of these gaps will be filled in next year through local and regional surveys which are planned with the assist-

1

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

ance of the WHO/FAO brucellosis centers now being established in various countries. AN INTERNATIONAL APPROACH

Until recently, mass inoculation campaigns and quarantine regulations were almost the sole forms of international action against communicable diseases. WHO and FAO have gone much further in their efforts in the international control in man and animals. These agencies are concerned with the epidemiology and epizootiology of diseases in the widest sense, as embracing the entire disease complex and the social and ecological factors involved. Brucellosis provides a challenging problem with respect to this concept of international action, in that an effective approach to the disease requires adaptation to various conditions and economies. Fortunately, however, for the convenient prosecution' of a world-wide program, the major aspects of brucellosis are of immediate importance to both advanced and undeveloped countries. I have considered in another paper" the main needs and problems encountered in brucellosis at the present time, but a brief recapitulation of a few of them might be helpful in evaluating, from an international viewpoint, what has already been done and what steps are indicated for the future. The needs and problems may be summarized as follows: (a) Surveys on the prevalence of the disease in man and animals, and the improved reporting of statistics. (b) The relative importance of contact exposure versus ingestion of infected food products in the transmission of brucellosis in different localities. (c) The importance of brucellosis as a cause of abortion in women and as an infection of infants and children. (d) The international standardization of the sero-agglutination test; clarification of the limitations of this test and of the complement-fixation test, and their role in the diagnosis of brucellosis. (e) The improvement and standardization of intradermic diagnostic agents for man and animals. (f) Simplified bacteriological technique for cultural and typing purposes. (g) Vaccine and chemotherapy in human brucellosis. (h) The use and improvement of vaccines and other control techniques in brucellosis of livestock. The need for more accurate knowledge on the prevalence of brucellosis has been mentioned previously, and the advantage of periodic compilation and distribution of statistical reports by an international agency is evident. The problems indicated in (b) and (c) above are suitable for local investigations, perhaps through some of the brucellosis centers set up by WHO and FAO. In this connection, it would be desirable to carry out experiments on the persistence of Br. abortus and melitensis in meat and dairy products under the prevailing conditions of preparation and

BRUCELLOSIS-A WORLD PROBLEM

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consumption of these products in various localities. Reports on this subject are sufficiently meagre and conflicting to warrant further study. Perhaps the most acute need from the point of view of international co-ordination and agreement in the field of brucellosis is the standardization of biological products and laboratory techniques. A case in point is the sero-agglutination test, which will be discussed further in other papers at this Congress. Surveys have clearly demonstrated that there are wide variations in the results obtained from, and the techniques of performing, the sero-agglutination test in different medical and veterinary laboratories. These variations include antigens of unequal sensitivity, the use of different serum dilutions, and divergent interpretations as to what constitutes a positive titer. Some efforts have been made, particularly in the veterinary field, to standardize the sero-agglutination test. In 1937, for example, the International Office of Epizootics adopted a standard serum and recommended its use to member countries with the hope that a degree of uniformity in the test would be achieved in veterinary laboratories. This project was interrupted by the war and was resumed in 1948. At the last meeting of the Office in May, 1950, it was resolved to advance the standardization of this test by the use of standardized antigens. It is to be hoped that through this Congress, and through the WHO/ FAO brucellosis centers, an extension of the principle of standardization will be achieved so that results of sero-agglutination tests reported by different laboratories will have a similar basis of reference. The same principle should be applied, wherever possible, to the various intradermic diagnostic agents at present used in man, milk ring test antigens, and vaccines used in human therapy and for the prevention of brucellosis in animals. In any international approach to a disease problem, we must always keep before us the fact that we are dealing in large part with economically underdeveloped countries. If our efforts at disease control are to be successful in these countries, simplified procedures and techniques must be developed. Much remains to be done in this connection in the field of brucellosis. Thus, simplified bacteriological techniques in isolating and typing Brucella, and in the production of Strain 19 vaccine are very much needed if these measures are to be used on the widest possible scale. More effective vaccines and improved diagnostic techniques for sheep, goats and swine would be of great assistance in many countries. The new antibiotics, although far from being the ultimate in brucellosis therapy, are giving promising results in human beings, but they are beyond the economic reach of the majority of sufferers; for this reason alone a critical analysis of vaccine therapy and perhaps of other inexpensive therapeutic procedures would be worthwhile. The WVIO and FAO have attempted to meet some of the problems

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

discussed previously by organizing WHO/FAO brucellosis centers in a number of countries to serve the needs of various regions. The following centers have already been established: Veterinary Microbiological Institute Botanikos Athens, Greece Dr. Costas Melanidis, Director

State Veterinary Serum Institute Bülowsvej 27 Copenhagen, Denmark Dr. Axel Thomsen Chief of Brucellosis Department

University of Florence Institute of Hygiene Center for the Study of Brucellosis Viale G. B. Morgagni 48 Florence, Italy Prof. G. Mazzetti, Director

Centre de Recherches sur la Fievre Ondulante Institut Bouisson-Bertrand Rue de l'Ecole-de-Médecine Montpellier, France Prof. L. Carrére, Directeur

Onderstepoort Veterinary Laboratory Onderstepoort Union of South Africa

Brucellosis Centre State Laboratory of Hygiene Rijeka, Yugoslavia Dr. J. Srakocic, Chief, Brucellosis Department

Commonwealth Serum Laboratories Parkville N. 2 Victoria, Australia Dr. F. G. Morgan, Director Ministry of Agriculture and Fisheries Veterinary Laboratory New Haw, Weybridge Surrey, England Dr. A. W. Stableforth, Director

State Veterinary Institute Etilik, Ankara Turkey Dr. M. Z. Muslu, Director

Centers are planned for at least two South American countries; Mexico; United States, and Asia. The laboratories in these countries have not yet been selected. These centers are occupied in studies of the bacteriology, transmission, diagnosis and therapy of the disease. They also serve as teaching centers for clinical and laboratory workers of their own and nearby countries, and in their work, emphasis is placed on the aspects of brucellosis of most importance to their particular regions. Thus, the centers in France and Yugoslavia are investigating the use of intrapalpebral inoculations of a non-antigenic intradermal agent for the diagnosis of brucellosis in sheep and goats, and the possible application of the milk ring test for these animals; in Copenhagen, studies are in progress on the comparative efficacy and standardization of milk ring test antigens; and consideration is being given in other centers to the problem of agglutinin-blocking antibodies in the sero-agglutination test, and the simplification of Strain 19 production. Fellowships have been awarded to workers in various centers to study brucellosis techniques in countries other than

BRUCELLOSIS-A WORLD PROBLEM

13

their own, and financial assistance has been provided, where necessary, for the purchase of apparatus and materials. In addition, literature on the latest advance in brucellosis is circulated to these centers by WHO and FAO. A WHO/FAO Expert Panel on Brucellosis comprising medical and veterinary experts drawn from all parts of the world has been organized to formulate specific recommendations for the guidance of WHO and FAO in their present and future activity in brucellosis. Moreover, the WHO and FAO are collaborating with the International Office of Epizootics in this work. By these means the agencies of the United Nations Organization are endeavoring to bring about closer relationships between brucellosis workers everywhere, and thereby to achieve a concerted attack on this problem on a global basis. We welcome warmly the valuable contribution of the Inter-American Congresses in the fight against brucellosis. REFERENCES (1) Carrere, L., Directeur, Centre de Recherches sur la Fievre Ondulante, Montpellier, France: Personal communication to the author. (2) Mingle, C. K.: Correlation of research with brucellosis control. Vet. Med. S4: 255 (July) 1948. (3) WHO Epidemiological and Vital Statistics Report, 3: 110 (May) 1950. (4) Jour. Am. Med. Assoc. editorial, 134: 876 (July 5) 1947. Evans, Alice C.: Brucellosis in the United States. Am. Jour. Pub. Health 37: 139 (Feb.) 1947. (5) Van Drimmelen, G.: The brucellosis survey in South Africa. Jour. S. Afr. Vet. Med. Assoc. 30: 178 (Dec.) 1949. (6) Polding, J. B.: Brucellosis in India. Ind. Jour. Vet. Sc. 17: 147, 1947. Abst. Vet. Bull. 19: 651 (Dec.) 1949. (7) Said Bilal Golem: L'Etat actuel des brucelloses en Turquie. Rev. turque d'Hyg. et Biol. Exp. 9: 63, 1949. Abst. Bull. Off. Int. Epiz., 33: 213 (Mar.-Apr.) 1950. (8) Holmberg, J.: Experiences in the control of contagious abortion in Finland. Proc. XIVth Int. Vet. Cong., London, 1949. (9) Verge, J.: La prophylaxie des brucelloses-Etat actuel de la question en médicine humaine et en médicine vétérinaire. Rev. Path. Comp. 50: 157 (Mars) 1950. (10) Buddle, M. B.: Vaccination against bovine brucellosis in New Zealand. Proc. XIVth Int. Vet. Cong., London, 1949. (11) Kaplan, M. M.: A Summary of the present status of brucellosis. Bull. World Hlth. Org. In press. LA BRUCELOSIS-PROBLEMA MUNDIAL (Sumario) En muchos países empieza a reconocerse que la brucelosis es una enfermedad a la cual no se le da la importancia que merece. La gravedad del problema en el Hemisferio Occidental es bien conocida. Se estima que en el continente europeo, exceptuando a los paises escandinavos, del 15 al 30% del ganado lechero está infectado. En aquellas partes del mundo adonde se han introducido razas de ganado europeo, tales como partes de Africa, India, China, Japón y Oceanfa, se ha presentado la brucelosis. La Brucella abortuses la especie que predomina en

lo

14

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

dichas zonas. Es interesante observar que la Br. suis aparentemente tiene poca significación fuera del Hemisferio Occidental, pero sobre este particular queda mucho por hacer. En Turquía, Grecia, Yugoeslavia, España y partes de Italia y Francia, la Br. melitensis es la causa principal de esta enfermedad en el hombre. A principios de 1950, la Organización Mundial de la Salud, a solicitud de las Naciones Miembros, comenzó a prestar ayuda en la coordinación de la campaña contra la brucelosis en el mundo. El trabajo se emprendió en cooperación con la Organización de las Naciones Unidas para la Agricultura y la Alimentación. Debido a la confusión que resulta de la multiplicidad de procedimientos empleados para el control de la enfermedad en el hombre y en los animales, muchos de ellos de muy dudoso valor, fué indicado que el problema de brucelosis debería ser enfrentado en forma internacional. La OMS y la FAO han tratado de resolver este problema designando catorce centros OMS/FAO para brucelosis en distintos países, destinados a satisfacer las necesidades de varias regiones. Estos centros han emprendido estudios sobre la epidemiología, epizootiologia, diagnóstico y terapia de la enfermedad; y sirven como centros de enseñanza para personal de las clínicas y laboratorios, locales y de otros paises. Hay becas disponibles para estudios. Por medio de estos centros la OMS y la FAO están también fomentando la estandardización de los procedimientos de laboratorio para el diagnóstico de la brucelosis en el hombre y en los animales, con el objeto de que, en cuanto sea posible, los estudios publicados sobre esta enfermedad en cualquier país, tengan una base común de referencia en otras partes. El Grupo de Peritos de la OMS y la FAO para la brucelosis celebrará sesiones en Washington durante e inmediatamente después de las sesiones del Tercer Congreso Interamericano. Se pedirá al grupo que formule recomendaciones específicas para el tratamiento y control de la brucelosis en el hombre y en los animales, y que indique pautas convenientes que puedan servir de orientación a la OMS y a la FAO. La OMS y la FAO han solicitado la colaboración de la Oficina Internacional de Epizootía y la del Comité Permanente de Congresos Internacionales de Veterinaria en la realización del programa de brucelosis. Se espera que por medio de estas organizaciones se puedan tomar medidas para limitar el tráfico internacional de animales infectados.

EPIDEMIOLOGIA DE LA BRUCELOSIS HUMANA EN LA REPúBLICA ARGENTINA II. LA BRUCELOSIS DEL ESTE Y DEL OESTE

Por E. A. MOLINELLI, E. M. FERNÁNDEZ ITHURRAT, D. ITHURRALDE, G. BASSO, S. MIYARA, A. SPERONI Y G. PANDOLFO Instituto Malbrán del Ministerio de Salud Pública de la Nación En la República Argentina la brucelosis del hombre está directamente vinculada a las enzoosis provocada en tres ganados domésticos (caprino, porcino y bovino)' por el parasitismo de microorganismos del género Brucella. Se ha demostrado que la enfermedad humana incide en el territorio comprendido entre los paralelos 22 y 46 de latitud sur, pero se carece de datos respecto de los residentes de la Patagonia austral (Santa Cruz, Tierra del Fuego e Islas Malvinas). A casi 3,500 enfermos asciende el número de brucelosos estudiados por nosotros con la cooperación del cuerpo médico nacional. Su edad oscila entre 8 meses y 82 años. Predominan netamente los adultos jóvenes, ya que algo más de la mitad corresponde a individuos de 21 a 40 años. En cuanto al sexo, la brucelosis humana indígena priva entre los varones-a éstos corresponden las tres cuartas partes de la morbilidad total -en razón de que, por la índole de las profesiones que afecta, la mayoría de las personas expuestas al contagio son hombres. El sexo masculino prevalece significativamente en los diferentes grupos de edades, excepto en menores de 10 años y en mayores de 70, entre los cuales la distribución es prácticamente igual en ambos sexos. Tanto si se trata de enfermedad profesional como si se debe a otro modo de contagio, la morbilidad estacional es mayor en primavera y verano y menor en otoño e invierno. Entre nosotros la brucelosis humana suele contraerse por: (1) contacto directo profesional con animales brucelosos y subproductos de éstos; (2) ingestión de alimentos infectados; (3) concomitancia de contacto profesional e ingestión de alimentos infectados; (4) permanencia en ambientes infectados o en la proximidad de los mismos; (5) infección accidental en laboratorios de bacteriología, y (6) contagio interhumano. La importancia porcentual de cada uno de estos mecanismos de infec1 Hasta ahora ni en los ovinos ni en los equinos de la Argentina ha sido aislada ninguna especie de Brucella. Los estudios existentes o arrojan resultados negativos (Sautu Riestra, 1943; Manzullo, 1949, etc. en ovinos) o sólo relatan la comprobación de bajos títulos aglutinantes en pocas cabezas de estos ganados (Pardal, 1933; Quevedo, 1939; Harispe y Ruiz, 1945; Minoprio, 1947; etc.) 15

16

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

ción brucelosa indígena, es la siguiente:

Fruencrwa %

Contacto directo profesional con animales brucelosos ................ Ingestión de alimentos infectados ..................................... Contacto profesional e ingestión de alimentos infectados .............. Permanencia en ambientes infectados por brucelas o en su proximidad Infección de laboratorio .............................................. Interhumano (lactancia materna, coito) ..............................

42.0 22.0 31.0 4.0 0.6 0.2

Los casos de brucelosis por contagio directo profesional con animales brucelosos se registran entre: (a) individuos dedicados a la faena, industrialización y transporte de reses para el consumo (personal de mataderos y frigoríficos que sacrifican ganados o elaboran subproductos animales; técnicos de la inspección veterinaria; personal ocupado en el transporte y recepción de hacienda y visceras; carniceros, etc.); (b) individuos que crían y explotan ganados (cabreros, tamberos, mayordomos y peones de campo, veterinarios, hacendados, granjeros, criadores de cerdos, etc.) en el curso de diversas tareas pecuarias (cuidar, ordeñar, desollar, etc., animales brucelosos; asistir vacas en trance de parición; extraer a mano restos de placenta; manipular fetos, etc.), y (c) individuos que manipulan tripas, cebo, o elaboran chacinados; obreros de barracas o curtiembres en contacto con cueros frescos o salados, etc. En estos casos (a, b y c), aunque aparentemente la única puerta de entrada es la piel, intacta, fisurada o con soluciones accidentales de continuidad es imposible excluir la probable contaminación por brucelas a través de las mucosas ocular, nasal, labial y bucal. En cambio, por la mucosa de revestimiento del tubo digestivo2 han

contraído la brucelosis aquellas personas que entre nosotros registran como único antecedente epidemiológico la ingestión de alimentos infectados de origen animal, a saber: leche cruda de vaca y cabra, queso fresco de vaca, queso fresco-'quesillo" (requesón y naterón)-y queso estacionado de cabra; cremas; "ricota" y manteca sin pasteurizar; carne y vísceras de cabrito insuficientemente cocidas. Además, en el centrooeste argentino el agua acumulada en pequeñas represas rurales ("aguadas"), de los puestos de cabras especialmente, debe ser considerada en potencia como fuente de infección brucelosa humana, pues esta agua estancada satisface simultáneamente las necesidades de hombres y animales. En la nosología argentina, la brucelosis por ingestión de leche cruda 2 En este caso se habla genéricamente, por no saberse aún a ciencia cierta en cuál de sus tres sectores (ingestivo, digestivo y eyectivo) tiene lugar la penetración de las brucelas. Sin embargo, el documentado trabajo experimental de U. Pagnini (Gior. Batt. Imm. 1939, 22, (4): 595-617), nos inclina a sostener también para el hombre, la importancia predominante de la mucosa bucofaringea como puerta de entrada de la infección brucelosa, en condiciones naturales y en cuanto al aparato digestivo se refiere.

BRUCELLOSIS HUMANA EN ARGENTINA

17

de vaca no tiene la importancia epidemiológica que reviste en otros países, dada la feliz circunstancia de estar muy arraigada la costumbre de hervir este alimento antes de ingerirlo. La mayor parte de los casos en que se registra el antecedente de consumir leche cruda de vaca, acaece entre los miembros de la colectividad anglosajona o entre personas que por considerar de mejor calidad supuestas "leches pasteurizadas", las ingieren sin someterlas previamente a la ebullición. El consumo exclusivo de otros lacticinios de vaca, tampoco tiene importancia estadística en la determinación de enfermos de brucelosis. Dentro de su escasa incidencia, merece agregarse el de un catador de cremas destinadas a la elaboración industrial de manteca, el cual-por más que sea obvio decirlo-tan sólo gustaba las muestras sometidas a su examen. Contrariamente, alto valor epidemiológico tiene la ingestión de lacticinios de cabra, en particular el denominado "quesillo", alimento muy difundido en el centro, oeste y Patagonia argentinos. Como casos excepcionales citaremos el de dos lactantes que adquirieron brucelosis al nutrirse con leche materna. Ambas madres padecían la noxa y hallábanse en pleno periodo de estado. De una de las leches se aisló Br. melitensis. En cambio, cuando el contagio se establece por permanencia en ambientes contaminados (casi siempre motivada por razones profesionales) o en sus proximidades, entonces sí puede afirmarse categóricamente que son puertas de entrada las mucosas ocular, nasal y labial y que el mecanismo de infección ha sido respectivamente facilitado por la adhesión, inhalación y deglución de partículas infecciosas. A la circunstancia de permanecer en ambientes infectados deben imputarse los casos de brucelosis que hemos registrado entre el personal de mataderos y frigoríficos de carnes sin contacto directo con reses o derivados (v.g., jefe y obreros de la sección mecánica, jefes administrativos y oficinistas, servicio médico del establecimiento, policía interna, ordenanzas, choferes, albañiles, foguistas, ascensoristas, pintores, carpinteros, electricistas, tacheros, hojalateros, vendedor de diarios, etc.) A la contingencia de la simple estadía, breve o prolongada, en las proximidades de un ambiente contaminado, cabe atribuir el caso del bruceloso cuyo único antecedente epidemiológico fehaciente era habitar, calle por medio, frente a una plaza ferroviaria de estacionamiento de ganado; a aquellos ocurridos entre rematadores de hacienda, etc. Y lo mismo puede decirse de otros casos singulares. Nos referimos, por ejemplo, a quienes han contraído brucelosis sin más antecedente probado que el de: (1) haber concurrido a "puestos" de cabras; (2) estar dedicados al comercio de cabras o de guano; (3) ser jardinero en un mercado de hacienda en pie; (4) visitar circunstancialmente un laboratorio en momento en que se filtraban varios litros de cultivo de Br. melitensis (de

18

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

la señorita protagonista de este último caso, pocas semanas después-por hemocultivo-se aislaba Br. melitensis).3 Según lo establece nuestra encuesta epidemiológica, la brucelosis profesional de los técnicos que trabajan en laboratorios de bacteriología, contraída por infección accidental, es debida casi siempre a la manipulación de cavias inoculados con materiales brucelosos y la subsecuente adhesión y deglución de polvo atmosférico cargado de detritus de estos animales de experimentación. La infección accidental de laboratorio ocurrió en uno de los autores (E. M. F. I.), a pesar de manipular los cavias con guantes de goma que protegían sus brazos hasta los codos. En la brucelosis contraida por contacto sexual, el agente etiológico llega al organismo por la mucosa genital. Nuestra casuística, que comprende dos mujeres esposas de brucelosos en actividad, incide muy pobremente en el total de enfermos estudiados. En cuanto al posible mecanismo potencial de la infección brucelosa interhumana por efecto de la transfusión de sangre (dadores individuales o material de "bancos de sangre"), ni en nuestro historial clínico ni en la bibliografía nacional, se registra caso fehaciente alguno. Ya reseñados los distintos mecanismos de la infección brucelosa indigena, es oportuno decir que cada una de estas modalidades de infección gana o pierde importancia epidemiológica en las diferentes regiones geográficas donde incide la brucelosis, y dentro de estas últimas, de acuerdo a la condición de vida, urbana o rural, de sus habitantes. Así, en las ciudades y pueblos del litoral argentino, donde están instalados los grandes mataderos y frigoríficos de carnes, la brucelosis se contrae generalmente por contacto directo profesional con animales brucelosos o subproductos, y la permanencia en ambientes contaminados por brucelas; en tanto que, en las ciudades y villas de las regiones andina y central, la noxa se adquire por ingestión de lacticinios infectados (preferentemente por consumo del "quesillo" de cabra). A mayor abundamiento, cabe agregar que en los grandes centros urbanos del litoral (Capital Federal, Rosario, Avellaneda, Zárate, Berisso, Colón y Valentín Alsina) donde no se crían ni se sacrifican caprinos en cantidades significativas, el 90% de los casos de brucelosis con comprobación bacteriológica, ocurre entre el personal de los mataderos y frigoríficos de carne, donde se faena exclusivamente hacienda bovina, porcina y ovina. En los ambientes rurales argentinos la modalidad prevaleciente es el contacto directo profesional con animales brucelosos; sobre todo en el cuidado, cría, explotación y comercio de los ganados vacuno y porcino, en la región litoral, y del ganado caprino en el centro, región andina y Patagonia. En estas tres últimas zonas, se agrega la ingestión de leche ' Una contaminación similar ocurrió en dos técnicos del Instituto Malbrán, los cuales sólo presenciaron el filtrado de cultivos para elaborar melitina.

BRUCELLOSIS HUMANA EN ARGENTINA

19

y lacticinios de cabra y el consumo de carne y vísceras de cabrito, insuficientemente cocidas. Si a lo expuesto anteriormente se agrega que las tres cuartas partes de los enfermos estudiados por los autores han contraído la enfermedad TABLA I.-Confrontación de los datos de la encuesta epidemiológica con la especie

de Brucella aislada en 800 casos humanos de brucelosis de la Argentina Fuente de Infección

1

2

3

4

5

6 7 8 9 10 11

Caprinos y/o ingestión de leche cruda de cabra y/o ingestión de lacticinios de cabra.............. Idem + otros ganados (bovinos, procinos u ovinos) con/sin ingestión de leche cruda de cabra o vaca y/o ingestión de lacticinios de cabra y/o vaca .............. Ambientes donde hay ganado caprino con infección brucelosa (sin contacto directo con estos animales) ........................... Bovinos y/o ingestión de leche cruda de vaca y/o ingestión de lacticinios de vaca.......... (A-a) Idem + otros ganados (porcinos u ovinos) con/sin ingestión de lacticinios de vaca............ (B-b) Porcinos ..................... (C-c) Idem + ovinos ................ (D) Ovinos ......................... (E) Ambientes infectados de mataderos o frigoríficos de carne*........(F) Infecciones de laboratorio ......... Contagio interhumano .............

Br. melitensis

272

Br. abortus Bru Bzr.sis Br. cortus

-

86

5

Buel Brucella atípica

1

-

2

-

1

-

-

-

11

91

111

-

3 1

46 62 6 21

20 5 2 1

-

1 2 1

30 3 -

10 2 -

-

382

261

152

-

3

1

5

* Los enfermos incluidos en el renglón 9 son aquellos que por razones de trabajo (mecánicos, electricistas, albañiles, pintores, carpinteros, oficinistas, sección vigilancia, foguistas, etc.) frecuentan los distintos ambientes de recepción de hacienda, matanza y elaboración de carnes de estos establecimientos, sin realizar tareas que exigen contacto directo con animales o subproductos.

durante el ejercicio de su trabajo u oficio, emerge entonces, con toda claridad, la extraordinaria importancia que en la Argentina tiene la brucelosis profesional. A los efectos de establecer la relación entre fuente de infección y especie de Brucella infectante, se han confeccionado las Tablas I, II y III. En la Tabla I hállanse confrontados los datos de la encuesta epidemiológica con el resultado de la clasificación bacteriológica correspondiente a 800

20

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

casos humanos de la Argentina, en los cuales los autores aislaron la Brucella causal. En las Tablas II y III, tales datos han sido discriminados para facilitar la mejor comprensión de su signifcado, según correspondan o no a casos infectados en mataderos y frigoríficos de carnes. La lectura de la Tabla I demuestra que cuando la brucelosis humana depende exclusivamente de la: (1) enzoosis caprina, siempre se aisla Br. melitensis (Tabla I, renglón 1); (2) enzoosis bovina, se aisla en orden decreciente de importancia Br. abortus, Br. suis y Br. melitensis (Tabla I, renglón 4); (3) enzoosis porcina, se aisla casi siempre Br. suis, en pocos casos Br. abortus y excepcionalmente Br. melitensis (Tabla I, renglón 6). TABLA II.-Discriminaciónde 328 brucelosos de la tablaI, los cuales contrajeronsu enfermedad trabajandoen mataderos o frigoríficos de carnes y derivados* Fuente de infección

A B C D E F

Br

Bovinos ......................... Idem + porcinos y/u ovinos ..... Porcinos ................... Idem + ovinos .................. Ovinos ................... ....... Ambientes infectados de estos establecimientost................

B s m¢litensis

1 1 1...... 1

r.

is

aborBr. t atípica ca

86 44 58 6 21

45 16 2 2 1

-

1

30

10

1

4

245

76

3

2 -

* Una parte de los enfermos correspondientes a los renglones A-E tienen contanto con animales en pie, pero la gran mayoria trabaja con las carnes y subproductos de la especie animal indicada en cada renglón. t Véase llamada (*) de la Tabla I.

Por tanto, los datos precedentes ponen de manifiesto que también en nuestro territorio hay intercontaminación por brucelas, que afecta a los ganados bovino (con Br. suis y probablemente Br. melitensis) y porcino (con Br. abortus).4 La intercontaminación del ganado vacuno con Br. suis resulta muy elevada, de atenernos a los datos de la Tabla II, renglón A, o sea calculando la frecuencia (65%) de aislamiento de Br. suis entre los individuos que contraen brucelosis manipulando reses vacunas y subproductos en los mataderos y frigorificos de carne. En cambio, entre los individuos que se contagian de brucelosis por ingerir leche cruda o lacticinios de vaca, o trabajar con ganado vacuno fuera del ambiente de mataderos 4 Desde el punto de vista bacteriológico cabe consignar que: (1) En regiones argentinas carentes de cabras hemos aislado Br. melitensis de enfermos con antecedentes de contacto profesional con vacas o de ingerir lacticinios crudos de vacas; (2) Fernández Ithurrat y Molinelli (1934) y Cedro (1945) han aislado Br. suis de muestras de leche de vacas de tambos de la ciudad de Buenos Aires y partidos circunvecinos; (3) Aun no se ha aislado Br. melitensis de bovinos.

21

BRUCELLOSIS HUMANA EN ARGENTINA

y frigoríficos de carne, la frecuencia del aislamiento de Br. suis se reduce al 7% (Tabla III, renglón a). En cuanto a los 22 brucelosos cuyo antecedente de contacto profesional señala al ganado ovino como única fuente de infección (Tabla II, renglón E), cabe destacar que todos ellos eran obreros que trabajaban en plazas de matanza de frigoríficos de carne, situadas justamente en un sector contiguo y dentro del mismo ambiente destinado al sacrificio de hacienda porcina. Esto explica la aparente discrepancia entre este dato (Tabla TABLA III.-Discriminación de 472 brucelosos de la Tabla I, infectados en zonas rurales o urbanas sin conexión con mataderos of frigorificos de carne Fuente de Infección

Caprinos y/o ingestión de leche cruda y/o lacticinios de cabra ........... (1) Idem + otros ganados (bovinos, porcinos u ovinos) con/sin ingestión de leche cruda y/o lacticinios de cabra y/o lacticinios de cabra y/o vaca ......... (2) Permanencia en ambientes donde hay ganado caprino con infección brucelosa (sin contacto directo con estos animales) ............................ (3) a) Bovinos y/o ingestión de leche cruda y/o lacticinios de vaca ............. (4) b) Idem + otros ganados (porcinos u ovinos) con/sin ingestión de leche cruda y/o lacticinios de vaca ....... (5) c) Porcinos .......................... (6) Infecciones de laboratorio ........... (10) Contagio interhumano .............. (11)

Br meitensis

Br. suis

272

--

86

2

5

Br. abortus

1

-

1

-

-

10

5

66

2

2 4 3

4 3 2

2 1 378

Brucela atipica

-

-

16

76

1

-

2

II, renglón E) y la inexistencia de infección brucelosa en el ganado ovino en la Argentina.' Finalmente, la Tabla II permite establecer que los ambientes de mataderos y frigoríficos de carnes están altamente contaminados con Br. suis, como lo demuestra particularmente el renglón F, pues entre 42 enfermos, sin antecedentes de contacto con reses o subproductos (personal de la sección mecánica, policía interna, oficinistas, electricistas, albañiles, pintores, foguistas, carpinteros, etc.), 30 estaban infectados por esta especie de Brucella. Por otra parte, en ciertos brucelosos de nuestro país, la encuesta epidemiológica señala a más de una especie animal como fuente de origen de la noxa. En estos casos, cuando entre ellas figuran los caprinos (Tabla I, renglón 2), en el 97% de los enfermos se aisló Br. melitensis; y cuando

22

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

figuran los bovinos y porcinos (Tabla I, renglón 5), en el 71% de los casos se aisla de los enfermos Br. suis. Estos resultados sugieren la mayor importancia que en la Argentina tiene Br. melitensis y Br. suis como especies causales de la brucelosis del hombre. II. LA BRUCELOSIS DEL ESTE Y DEL OESTE

El estudio epidemiológico, bacteriológico y clínico de esta zoonosis, permite fundamentar el concepto de la existencia en la República Argentina de dos grandes grupos nosográficos: (1) La brucelosis del Este y (2) La brucelosis del Oeste, que, en t.érminos latos, encuentran en la enzoosis bovino-porcina y en la enzoosis caprina, su respectiva fuente de infección. La brucelosis del Este incide entre los habitantes que viven en todo el territorio de la Capital Federal, provincias de Buenos Aires, Santa Fe, Entre Ríos, Corrientes, gobernación de Misiones, parte oriental de La Pampa, planicie de Córdoba, departamento Rivadavia de Santiago del Estero y parte oriental de las gobernaciones de Chaco y Formosa. La brucelosis del Oeste afecta a pobladores de las provincias de Jujuy, Salta, Catamarca, La Rioja, San Juan, Mendoza, San Luis, Tucumán, la casi totalidad de Santiago del Estero, noroeste de Córdoba, centro y oeste de La Pampa, oeste del Chaco y Formosa y los territorios de Río Negro, Neuquén y Chubut. La autenticidad de ambos grupos está sólidamente corroborada por el resultado del estudio bacteriológico hecho en el Instituto Malbrán. Así, la Tabla IV demuestra la frecuencia preponderante de Br. suis y Br. abortus en los enfermos del Este y la presencia casi exclusiva de Br. melitensis en los del Oeste. Allí donde la enfermedad se origina por la infección de los ganados bovino y porcino, lo habitual es comprobar casos singulares de la noxa en cada hogar. En cambio, en las zonas de infección caprina, lo frecuente es observar varios miembros de una misma familia simultáneamente atacados de brucelosis en actividad. La enfermedad humana en la Argentina reviste, en general, una sintomatología y evolución diferente según se trate de casos originados en las regiones del este o del oeste del país. En el oeste, donde los casos humanos se deben a Br. melitensis, la enfermedad es más grave (antes del advenimiento de la aureomicina, cloromicetina y terramicina el promedio de duración de la enfermedad era de ocho a diez meses y la mortalidad arrojaba una cifra próxima al 8%); la fiebre adquiere casi uniformemente el tipo ondulatorio; la sintomatología nerviosa, digestiva (hepática, especialmente) y articular es más severa; el curso de la dolencia es poco influenciado por la vacunoterapia específica y son más frecuentes las recaídas después de administrar los antibióticos precitados.

23

BRUCELLOSIS HUMANA EN ARGENTINA

En el este, donde la enfermedad se debe principalmente a Br. abortus entre los individuos sin contacto con ganados y a Br. suis entre el personal de mataderos y frigoríficos de carnes, los casos son comparativamente menos graves; tanto por la escasa mortalidad que arrojan (alrededor del 2%), como por su más reducida duración (promedio cuatro a seis meses); la tendencia a la recuperación espontánea y los frecuentes éxitos terapéuticos, sin recaídas, obtenidos mediante la medicación por "shock" específico (utilizando la vacunoterapia por vía endovenosa) o la administración de los modernos antibióticos antes mencionados. TABLA IV.-Especie de Brucella aisladaen 800 enfermos de la Argentina y

distribución geográfica de los casos Región geográfica

Br. abortus

Br. suis

Br. melitensis

Brucella atípica

Total

Este .......... Oeste .........

148 4

260 1

14 368

4 1

426 374

152

261

382

5

800

Finalmente, desde el punto de vista de la temperatura corporal, la diferencia entre los brucelosos del este y del oeste se aprecia principalmente en el trazado de la curva termométrica. Los primeros muestran a menudo un brote febril único, en lugar de las clásicas ondas febriles, que caracterizan a los brucelosos de la zona oeste. BIBLIOGRAFÍA Amuchategui, S. R.; Villafafie Lastra, T. de; Goobar, J. K.; Elkeles, G.; Tey, J. A. y Schwartz, R.: Endemogeografia humana y animal de la brucelosis. Rev. Méd. de Córdoba, 35, (10): 523-528, 1947. Aribazalo, J. B. y Pomina, C. P.: La fiebre ondulante en los territorios de Rio Negro y Neuquén (República Argentina). Sem. Méd., 1, (17): 1442-1444, 1933. Atlas, J.: Epidemiología de la brucelosis en la provincia de Santiago del Estero. Dia MEdico, 19, (54): 1732-1736, 1947. Basso, G.; Miyara, S. y Molinelli, E. A.: La brucelosis en la provincia de Mendoza. Sem. Méd., 2, (28), 126-133. Addenda et Corrigenda, (29): 216, 1943. Basso, G. y Miyara, S.: Brucelosis en las provincias de Cuyo. Rev. Asoc. Méd. Arg., 62, (625-626): 119-125, 1948. Cedro, V. C. F.: Eliminación de Brucella por la leche de vacas reactoras. Rev. Méd. Cien. Af., 10, (12): 755-764, 1948 y 11, (1-3): 1-16, 1949. Cortelezzi, E. D.: Consideraciones epidemiológicas sobre brucelosis. Sem. Méd., 1, (8), 365-371, 1945; Dia Méd., 17, (38): 1062-1068, 1945; Anales de la Segunda Convención de los Médicos de la Industria, 1946, 105-119. Buenos Aires, Instituto Argentino de Seguridad, editor. Crescentino, H. H.: La fiebre ondulante en la provincia de San Juan. Semana Méd. 2, (36): 712-715, 1933. Rev. Méd. de Cuyo, 9, (84): 249-251. 1934. Criscuolo, E.: Epidemiología y tratamiento de la brucelosis. Rev. Méd. Córdoba, 33, (9): 657-659, 1945.

24

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Criscuolo, E.: Indice de infección brucelosa en la provincia de Córdoba. Rev. Méd. Córdoba, S4, (1): 3-12, 1946. Rev. San. Mil., 45, (3): 376-383, 1946. Fernández Ithurrat, E. M.: Mis investigaciones sobre las Brucellas. Rev. Méd. Vet., 23, (9-10): 366-377, 1941. Harispe, C. M. y Ruiz, G.: Indice de morbilidad de la brucelosis. La infección en la especia ovina. Anuario de la Facultad de Medicina Veterinaria, La Plata, 8, 67-80, 1945. Manzullo, A.: La infección brucelosa de los ganados ovinos de la República Argentina. Datos relativos a la Gobernación Maritima de Tierra del Fuego. Primera Reunión Panamericana de Enfermedad de Chagas, Primera Reunión conjunta de la Sociedad Argentina de Patología Infecciosa y Epidemiología y de la Sociedad Argentina de Patología y Epidemiología de Enfermedades Transmisibles. Tucumán, Salta y Jujuy, julio 11-16, 1949 (actas inéditas). Comunicación personal, octubre de 1950. Mazza, S.: Un foco antiguo del país y el más intenso de brucelosis de origen caprino en la provincia de Tucumán. VIII Reunión de la Sociedad Argentina de Patologia Regional del Norte, Santiago del Estero, 2 y 3 de octubre de 1933, 2: 825-829. Buenos Aires: Imprenta de la Universidad, 1934. Mazza, S. y Basualdo, C.: Foco de fiebre ondulante caprina en Angaco Sud (San Juan). IX Reunión de la Sociedad Argentina de Patologia Regional, Mendoza, octubre 1-4 de 1935, S, 1814-1815. Buenos Aires: Imprenta de la Universidad, 1939. Mazza, S. y Canal Feijoo, E. J.: Foco de fiebre ondulante en el departamento de Choya y reacciones de Wright de animales de la provincia de Santiago del Estero. Publicación No. 12 de la Misión de Estudios de Patología Regional Argentina. Buenos Aires: Imprenta de la Universidad, 1933. Mazza, S.; Caro, A. y Cores, L. B. de: Comprobación de focos de fiebre ondulante en los departamentos de Metán, Anta y Rosario de la Frontera, provincia de Salta. Publicación No. 10 de la Misión de Estudios de Patologia Regional Argentina. Buenos Aires: Imprenta de la Universidad, 1933. Mazza, S. y Giménez, A. V.: Un afo de observación de fiebre ondulante en el departamento de Santa Maria, provincia de Catamarca. Publicación No. 11 de la Misión de Estudios de Patología Regional Argentina. Buenos Aires: Imprenta de la Universidad, 1933. Mazza, S. y Herrera, J. Ch.: Observaciones de focos de fiebre ondulante en Andalgalá (Catamarca). IX Reunión de la Sociedad Argentina de Patologia Regional, Mendoza, octubre 1-4 de 1935, 3, 1815-1824. Buenos Aires: Imprenta de la Universidad, 1939. Mazza, S. y Herrera, J. Ch: La brucelosis en el Departamento Andalgalá de la Provincia de Catamarca. Pren. Méd. Arg., 28, (41): 1963-1965, 1942. Mazza, S. y Lovaglio, J. A.: Brucelosis en el Dep. de Cafayate, provincia de Salta y regiones vecinas. IX Reunión de la Sociedad Argentina de Patalogia Regional, Mendoza, octubre 1-4 de 1935, 3, 1780-1802. Buenos Aires: Imprenta de la Universidad, 1939. Mazza, S. y Rebosolan, J. B.: Observaciones de brucelosis en los departamentos de Choya, Guasayán y Moreno (Sgo. del Estero) IX Reunión de la Sociedad Argentina de Patologia Regional, Mendoza, octubre 1-4 de 1935, 3, 1758-1763. Buenos Aires: Imprenta de la Universidad, 1939. Mazza, S. y Ruchelli, A. P.: Un año de observación de la fiebre ondulante en los departamentos de Belén y Tinogasta, provincia de Catamarca. Publicación No. 11 de la Misión de Estudios de Patología Regional Argentina, Buenos Aires: Imprenta de la Universidad, 1933. Mazza, S. y Ruchelli, A.: Observaciones de brucelosis en el departamento Tino-

BRUCELLOSIS HUMANA EN ARGENTINA

25

gasta, provincia de Catamarca. IX Reunión de la Sociedad Argentina de Patología Regional. Mendoza, 1 al 4 de octubre de 1935, 3, 1828-1843. Buenos Aires: Imprenta de la Universidad, 1939. Mazza, S., Ruchelli, A. y Arroyabe, V.: Comprobación de focos de fiebre ondulante en la provincia de La Rioja. Publicación No. 10. Misión de Estudios de Patologia Regional Argentina. Buenos Aires: Imprenta de la Universidad, 1933. Mazza, S.: Santillán, P. y Gutdeutsch, H.: Sobre focos de fiebre ondulante en la provincia de Tucumán y regiones limítrofes. Publicación No. 6, de la Misión de Estudios de Patología Regional Argentina. Buenos Aires: Imprenta de la Universidad, 1932. Pren. Méd. Arg., 19, (21): 1306-1312, 1932. Minoprio, J. L.: Nota sobre infección brucelósica en ovinos. Haumania, 1, (1): 42-44, 1947. Molinelli, E. A.: Infección por Brucella en los frigoríficos y mataderos de la República Argentina. Sem. Méd. 1, (14): 1176-1179, 1933 a. Molinelli, E. A.: Estudio de la infección profesional por Brucella en ambientes urbanos de la República Argentina. Sem. Méd., 2, (50): 19191923, 1933 b. Molinelli, E. A.: La infección profesional por Brucella en algunos ambientes urbanos y rurales de la República Argentina. Rev. Asoc. Méd. Arg., 48, (337): 863-884, 1934. Sem. Méd., 2, (43), 1248-1258, 1934. Molinelli, E. A.: Misión sanitaria a Tinogasta (Prov. de Catamarca) con especial referencia a la brucelosis autóctona. Bol. San. Dep. Nac. Hig., 1, (4): 293-300, 1937. Molinelli, E. A.: Basso, G. y Miyara, S.: Epidemiología de la brucelosis humana en la República Argentina. Rev. Asoc. Méd. Arg., 61, (601-602): 182188. Primera Reunión Interamericana de la Brucelosis, México, 28 de octubre al 3 de noviembre de 1946: 43-61. Ediciones del Hospital General, Secretaría de la Salubridad y Asistencia, México, D. F., 1948, editor. Molinelli, E. A. y Fernández Ithurrat, E. M.: Estudio de la infección por Brucella en las vacas de los tambos de la ciudad de Buenos Aires. Sem. Méd., 2, (29): 176-186. Rev. Méd. Vet., 16, (2): 13-36, 1934. Molinelli, E. A.; Fernández Ithurrat, E. M.; Basso, G.; Miyara, S. y Fernández Ithurrat, M. C.: A survey of brucellosis in the Argentine Republic. Part I: Introduction. Part II: Clinical study. Part III: Biological diagnosis. Part IV: Confrontation between autochthonous cases of the west and littoral regions. Part V: Occupational brucellosis. Proceedings of the Sixth Pacific Science Congress (held at Berkeley, Stanford and San Francisco, July 24 to August 12, 1939), 5, 267-291. Berkeley, California: University of California Press, 1942. Molinelli, E. A.; Fernández Ithurrat, E. M. e Ithurralde, D.: Epidemiologia de la brucelosis humana en la República Argentina. Rev. Asoc. Méd. Arg., 62, (625-626): 111-119, 1948. Molinelli, E. A.: Fernández Ithurrat, E. M.; Basso, G.; Miyara, S.; Fernández Ithurrat, M. C.; Piorsky, I.; Zuccarini, J.; Ithurralde, D.; Gambino Rosa, V. de; Cornejo Saravia, E.; Burlando, A. J.; Iacapraro, G.; Insausti, T.; Malbrán, J.; Dambrosi, R. G.; Royer, M.; Thompson, V. E. R.; Pandolfo, G. P.; D'Alessandro, N. V. y Gambino, L. R.: Brucelosis humana en la República Argentina. Memoria del Primer Congreso Nacional de la Brucelosis, Montevideo, diciembre 15-17, 1947: 439-491. Montevideo: Imprenta Letras, editor, 1948. Panzeri, A.: Brucelosis en Serrezuela. Rev. Méd. Córdoba, 35, (11): 611-615, 1947.

26

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Pardal, E.: Fiebre ondulante. Investigaciones epidemiológicas realizadas en la provincia de San Luis. Sem. Méd. 1, (7), 590-594, 1933. Pardal, E.: La Brucelosis. Investigación, morbilidad, distribución y epidemiologfa en la provincia de San Luis. Sem. Méd., 2, (50): 941-948, 1945. Quevedo, J. M. (h.): Papel de las brucelas en los equinos y lesiones atribuibles a su acción. Presentación de algunos casos. Rev. Méd. Vet., 21, (7-8): 298-306, 1939. Rosas Costa, G. y Petric, J.: Investigación sobre divulgación brucelósica entre el personal de la industria de la carne en la provincia de Entre Rios. Primera contribución. Bol. Inform. del Ministerio de Salud Púb. de Entre Ríos, 1, (6): 8-15, 1949. Rosas Costa, G. A. y Puskovich, F.: Investigación sobre divulgación brucelósica entre el personal de la industria de la carne en la provincia de Entre Rios (segunda contribución). Bol. Inform. del Ministerio de Salud Púb. de Entre Rios, 1, (12): 11-23, 1949. Ruchelli, A.: La fiebre ondulante en el noroeste de la provincia de Catamarca. Tesis No. 4809 de la Facultad de Ciencias Médicas de Buenos Aires. Buenos Aires: Imprenta de la Universidad, 1935. Sautu Riestra, M. R. de: La brucelosis en la inspección de carnes. Rev. Méd. Cienc. Af., 5, (12): 901-910, 1943.

EPIDEMIOLOGY OF HUMAN BRUCELLOSIS IN THE ARGENTINE REPUBLIC (Summary) In the Argentine Republic human brucellosis is connected with the incidence in goats, swine and bovines. Brucella organisms have not been isolated from sheep. Human brucellosis is encountered between the 22 and 46 degrees latitude, South. Seasonal morbidity is greater in spring and summer and lesser in autumn and winter. Out of 3,000 patients, more than half range between the ages of 21 and 40, males being prevalent, except in cases under 10 and over 70 years of age, where the number of male and female cases is about the same. The source of infection is as follows: Occupational contact with Brucella infected animals .......... 42% 22% Infected food ............................................ 31% Occupational contact and infected food ..................... 4% Exposure in places infected with Brucella or their proximity ..... 0.6% Laboratory infection ...................................... Human contact (coitus, breast feeding) ..................... 0.2% In large towns of the Argentine littoral 90% of brucellosis cases are found among slaughter house and meat packing plant workers while in the towns and villages of the Andean region the chief source of human infection is goat milk products, cheese principally. In rural areas, infection from occupational contact with bovines and swine is prevalent in the eastern littoral region, while infection from goats is chiefly observed in the central, Andean and Patagonian regions. The drinking of milk is not an important epidemiological factor as the habit of boiling milk is widespread.

BRUCELLOSIS HUMANA EN ARGENTINA

27

Epidemiological data and bacteriological tests show intercontamination with Brucella organisms between bovines (with Br. melitensis and Br. suis) and swine (with Br. abortus); this is not observed in goats. In Argentina the danger to man of exposure to animals infected with Brucella is, in decreasing order of importance, goats, swine, bovines. Human brucellosis in Argentina may be classified into two nosographic groups: (1) Brucellosis of the East and (2) Brucellosis of the West, each having marked epidemiological, bacteriological, clinical and therapeutic characteristics.

BRUCELLOSIS INCIDENCE IN THE UNITED STATES By JAMES H.

STEELE,

D.V.M., and L. OTIS EMIK, Ph.D.

Veterinary Director and Scientist (R), respectively, Communicable Disease Center, PublicHealth Service, Federal Security Agency, Atlanta, Georgia The annual incidence of brucellosis in the United States is represented by the number of new cases of the disease occurring in the country during a year. This figure will never be known with great accuracy under conditions now existing. The reported figures represent attacks of brucellosis which may be either primary, relapses, or exacerbations of chronic cases. Some of these non-primary attacks have never been reported as primary cases, others have. The available figures are more closely akin to prevalence than incidence. The best approximation to true incidence could be calculated if the rates and methods of infection of man from source animals and the prevalence in animals could be determined. Surveys made up to the present are neither adequate nor appropriate for making any national extrapolations. Where appropriate population figures on the occupational occurrence of the disease are available, as in certain states, occupational rates may be calculated on the basis of these data, and national estimates calculated from them. It is necessary to assume that the occupational rates are fairly constant from state to state and that the variability of rates between states used to calculate the standard rates is a measure of the statistical error. The complete accuracy of this estimated figure will depend also upon the correctness of the primary assumptions, the selection of proper populations, and the reliability of sources of reported attacks. It is also possible to make some estimate of a national figure on the basis of laboratory results. The degree of error will be high due to the incompleteness and variability of the data at hand. Essentially, the method is to determine the ratio of acceptable cases to positive serum agglutinations at each different titer level and to estimate cases from laboratory results. The year 1949 has been chosen for this investigation. SELECTION OF METHODS AND SOURCE DATA

The accuracy of any data on brucellosis depends upon many factors. Particularly, diagnosis and reporting of cases must be relatively accurate in any area to be used as a standard. Epidemiologic investigations should be relatively complete to provide the necessary classification of the individual case, as well as to act as a check on diagnosis and reporting. Minnesota meets these requirements well. Iowa, Illinois, and Wisconsin 28

BRUCELLOSIS INCIDENCE IN THE UNITED STATES

29

provide good information but certain details are lacking. These four states were selected as the source of data for this phase of the study. The other states contributing information have not been solicited to as great an extent. Information from previous investigations in Iowa (Jordan-1942, 1950; Jordan and Borts-1947) and in Minnesota (Magoffin, Kabler, Spink and Fleming-1949) demonstrated the magnitudes of various attack rates for specific occupations. High rates were found for packinghouse and rendering-plant workers, veterinarians, and animal production farmers; and intermediate rates for butchers, processors, stock buyers, and stock handlers. The attack rates for children, housewives, and the remainder of the population were relatively low. TABLE 1.-Brucellosis cases by occupation in Minnesota, Iowa, Illinois, and

Wisconsin for 1949 State

Wisconsin. Iowa ...... Illinois.... Minnesota.

Veter- Packing house inarjano workers

Other animal animal contact

kes14

2 9

19 28

7 13

4

47

3

am Farmers

115 173 176 141

ChilHouseuse ghi"-Other wives

36 51 124 33

14 19 14 20

Other know known

Subtotal

Un kown

47 65 74 41

240 358 388 289

23 116 61

240 281 504 350

Rates for any occupational group can be calculated if the number of cases and persons in the group can be adequately determined. Selection of specific occupation groups which are limited to the same occupational exposure hazards as the cases to which they will be related should provide maximum accuracy in the derived rates. The available data for attacks of brucellosis by occupation in Minnesota, Iowa, Illinois, and Wisconsin during 1949 are presented in Table 1. Bureau of the Census figures were available for packing-house workers, farmers, children, housewives, and the remainder of the population. The number of veterinarians in the United States was obtained from their national directory. Farmers were defined as those engaged in animal husbandry, including dairy and livestock farms, plus subsistence farms and general farming. Undoubtedly, farms are not perfectly split this way. Many agronomists will have a few animals around, and some subsistence farms will have none. Children's rates were considerably more constant when only rural-farm children were considered. The same was true for rural housewives. No population figures could be specifically determined for those workers such as butchers, rendering-plant workers, stock buyers and handlers, dairy workers, and others having contact with animals or their products. Since this group is rather intimately related to slaughter, for the most part, it appeared most appropriate to add these cases to those of packing-house

30

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

oB

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BRUCELLOSIS INCIDENCE IN THE UNITED STATES

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workers. By adding all packing-house workers, and those processing meat as well, into one population, the variability of the rate for this group of cases was reduced by 60%. The remaining cases of known occupation gave most stable rates in the rural population. The populations for these various occupation groups are listed in Table 2 for the four study states and for the entire country. The average rates for the four study states by occupation group and their standard deviation, calculated by the method given in Pearl (1941) are presented in Table 3. TABLE 3.-Brucellosis average attack rates by occupation for Minnesota, Iowa,

Illinois and Wisconsin for 1949 Occupation

group

.

Veterinarians ............................ Packing house plus other animal contact workers .............................. Animal farmers ......................... Rural housewives ...................... Rural farm children. Remainder (rural) .8.53

Average annual rates .. (100,000)

Standard deviation of rate (%)

950.6

28

295.2 72.8 16.0 6.87

22 15 24 9 14

The adjustment of numbers of reported cases on the basis of laboratory results is subject to large sources of error. This method may be fairly good for one particular purpose, namely, to estimate the number of cases which would have been discovered if epidemiologic investigation were pursued diligently on all serum agglutination tests positive at dilutions of 1:80 or greater. To obtain the ratio of cases to titers, the distribution of titers in culturally proven cases determined by Magoffin et al. (1949), was compared to the distribution of titers for the ten states in Table 4 listing number of positive agglutination tests. The ratio of Minnesota titers to those in the ten states and the ratio of culturally proven cases to total cases reported in Minnesota are necessary to complete the estimates for the ten states. RESULTS AND DISCUSSION Estimated numbers of cases were calculated for each occupation group and for each state simply by multiplying the rate by the population and taking the sum of the calculated cases. There were 10,709 estimated cases for the United States in 1949 as compared to 4,143 cases officially reported by the National Office of Vital Statistics. The standard deviations of the rates for the average specific population units of the four states, used to calculate the rates, vary from 9 to 28% as shown in Table 3. Extrapolating and combining the occupational groups gives a standard

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Equine brucellosis was detected by serological tests in animals that suffered from fistulous whithers and periodic ophthalmia, but it was found also in horses that did not show any evidence of either disease. A strain of Brucella abortus was recovered from a dog in one instance. Two out of 90 sheep showed anti-Brucella agglutinins in significant titers. For the sake of conciseness the data ware tabulated in Tables I-VI, and we shall present some general remarks on bovine brucellosis, the only type that has merited more attention. The tables and the detailed sheets from which they were derived showed that there are highly infected herds in regions where other herds are immune or present a low incidence rate. We think that this difference must be credited to two principal items. Firstly, the lack of any sort of prophylaxis in contaminated herds, a fact that gradually increases the number of infected animals in a single herd. Secondly, the relatively natural segregation of some herds due to the great expanses of land where they are raised, often limited rather by the very frequent topographical irregularities than by fences or restrictive measures to the dispersion of cattle. A fact that must be stressed in connection with the low rates in some regions is the type of cattle raising in practice in a large part of the country; the animals live in immense pasturages or tracts of land and are assembled sometimes only once a year; thus animal to animal infections have little chance of occurring. Moreover, the eliminated microorganisms on the ground are more easily destroyed by the natural physico-chemical agents, before they have the opportunity of infecting other animals. To support this fact it suffices to mention the high rates in dairy cattle from the States of Rio Grande do Sul, Rio de Janeiro, Sao Paulo and Pará, compared with the relatively low rates in the range or common cattle of the same regions. Prophylactic measures against animal brucellosis are hardly used in the country. Of course the importation of infected animals from other countries is forbidden. All animals to be shown at Federal or State exhibitions must be free from the disease, and a certificate of negative serological test must be presented when registering animals in the herdbooks; this is partly the reason for the low rate in selected cattle. As has been observed in other countries, serological surveys have given rise in recent years to a gradual spread of the infection beyond most of the primitive foci because as soon as the breeder knows that some of his animals are reactors he sends them to the public market where they may be bought by farmers whose herds do not have the disease. In the States of Sao Paulo and Rio de Janeiro official measures concerning the control of brucellosis in dairy cattle were recently introduced; the animals must be submitted to periodic blood tests and reac-

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68

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

tors are not allowed to continue as dairy animals. These tests showed that the disease is still prevalent in many dairy herds. In a very few instances the reactors have been segregated. Slaughtering following a positive test is very rare because no indemnity is paid; the farmers not realizing the importance of this measure for their herds, prefer to sell the reactors. Strain 19 vaccine has been used in calves, in some States, without any particular planning except in the State of Sao Paulo. As its use is increasing at present, we think that all data based upon serological reactions henceforth must be regarded with caution if it is not stated whether the animals were vaccinated or not. This is of special importance in the State of Sao Paulo where a large scale trial mass vaccination of cattle, including adults, is in progress, mainly on dairy cattle. The results obtained until now are fairly good, but the persistence of agglutinins may restrict future control measures based on blood tests. The abortion ring test is being slowly introduced as an auxiliary tool for detecting infected dairy animals, but it is still in the experimental stage. Human infection parallels the animal disease, obviously bovine and porcine in origin, and is observed mostly in regions where there is a large number of animals. The rare cases in which Br. melitensis was recovered were doubtful or were observed in persons who possibly had been infected outside the country. Several resolutions have been approved in successive Congresses of Veterinary Medicine recommending that studies on brucellosis be intensified, but even a general knowledge of the disease is far from being achieved. The importance of the disease, both as an economic and public health problem, is not generally realized. It is necessary to encourage and to support the work of the few groups who, for many years, have been doing free-lance studies of the disease. We think that the need for information, standardization of methods for the diagnosis, plans for the control of the disease adapted to each particular region of the country, and many other items concerned with human and animal brucellosis in Brazil indicate that the organization of a Center for the study of the disease is worthy of consideration. We hope that in the near future this Center will be a reality. LA BRUCELOSIS ANIMAL EN EL BRASIL (Sumario) Los datos sobre brucelosis bovina muestran que la enfermedad está diseminada en el pais, tanto en el ganado para el consumo como en el ganado lechero, en una proporción del 10 al 20%. La incidencia de la infección en los cerdos es más alta llegando hasta 30 ó 40%. Como es de suponer, la enfermedad ha sido más estudiada donde se encuentran las concentraciones más grandes de ganado, como en los estados de Rio Grande do Sul, Sáo Paulo, Minas Gerais y Rio de Janeiro.

LA BRUCELOSIS ANIMAL EN EL URUGUAY

69

El diagnóstico ha sido establecido mediante el aislamiento de microorganismos de la placenta, fetos o leche, pruebas de aglutinación y manifestaciones clfnicas.

La brucelosis caprina es rara y solamente se han encontrado casos esporádicos por medio de pruebas de aglutinación en focos de brucelosis bovina. Hasta el presente, esto no tiene ninguna importancia epidemiológica. La brucelosis equina y canina es también esporádica y algunos casos han sido descubiertos mediante pruebas de aglutinación en focos de brucelosis bovina. En un caso se aisló la Brucella abortus de un perro. No es posible afirmar si la infección se propagó de algunos focos existentes o si habla existido por muchos años sin ser descubierta. La evidencia, sin embargo, parece indicar que ya existía en los lugares donde ha sido observada hasta ahora, y en muchos otros desconocidos aún, porque se han descubierto focos nuevos cada vez que se han efectuado estudios serológicos en rebaños ubicados en lugares donde no parecia existir la enfermedad. La infección humana, indudablemente de origen bovino o porcino, es comparable a la enfermedad animal. Los pocos casos en que se aisló la Br. melitensis fueron dudosos o fueron de personas que posiblemente hablan contraído la infección fuera del país.

LA BRUCELOSIS ANIMAL EN EL URUGUAY Por B. SZYFRES, J. L. STELLA, W. ERRANDONEA, H. TRENCHI, D. ABARACóN, J. C. PIÑON y J. M. INFANTOZZI

Servicio de Brucelosis. Laboratorio de Biología Animal "Dr. Miguel C. Rubino", Dirección de Ganadería, Uruguay El Uruguay es un país eminentemente ganadero. Los productos animales, lana y carne, constituyen su principal renglón de exportación. El número de ganado bovino, en su predominancia de producción de carne, se calcula actualmente entre 7 y 8 millones y el de ovinos entre 25 y 28 millones de cabezas. La brucelosis es uno de los principales problemas sanitarios de nuestra ganadería. Por la merma que ocasiona en el procreo y en la producción de leche, la brucelosis repercute directamente sobre la riqueza básica del país. Este informe tiene por objeto dar a conocer el estado actual de la cuestión en el Uruguay. Reunimos en él los datos existentes sobre la difusión de la enfermedad y las medidas que ha adoptado el Poder Ejecutivo para contrarrestar su extensión y reducir los estragos causados por ese mal. Los resultados obtenidos hasta ahora no son satisfactorios. El problema de la profilaxis tendrá que ser reconsiderado en su conjunto, con el fin de proveer al país de un programa nacional de control de la enfermedad, en consonancia con los últimos adelantos científicos y las características de nuestro medio.

70

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS DATOS SOBRE LA DIFUSIóN DE LA BRUCELOSIS ANIMAL

Brucelosis bovina.-La infección a Br. abortus fué comprobada en el Uruguay, en el año 1928, por A. Cassamagnaghi (1). En 1932 y 1933, el Laboratorio de Investigaciones (actual Laboratorio de Biología Animal de la Dirección de Ganadería) hace un estudio sistemático, con el fin de conocer el grado de difusión de la enfermedad en los establecimientos lecheros del Departamento de Montevideo (2). Se ha practicado el diagnóstico en 1,790 animales lecheros pertenecientes a 224 tambos. En 116 de esos establecimientos, es decir en un 51.7% de los examinados, se han encontrado animales con reacciones positivas a la prueba de la aglutinación. De los 1,790 animales examinados, el 20.3% dieron reacciones positivas y el 14.7% reacciones dudosas. El Laboratorio de Biología Animal de la Dirección de Ganadería (4) dió a conocer los datos correspondientes a las pruebas efectuadas entre 1932 y 1947. Esos datos se refieren a 113,645 muestras de sangre de bovinos, procedentes de 1,161 establecimientos repartidos por la República. Los exámenes efectuados respondieron a distintas razones, tales como: estudio epizootiológico, diagnóstico por haberse producido abortos en el establecimiento de origen, saneamiento de establecimientos infectados, exportación, y desde 1942, el dar cumplimiento a la Reglamentación de Exposiciones, Ferias y Remates. El 31.9% de los 1,161 establecimientos, de los cuales hemos examinado muestras de sangre, con un promedio de 97 muestras por establecimiento, contenían animales positivos a la seroaglutinación. Las siguientes cifras indican el índice de animales infectados: Reacciones positivas ................................... Reacciones dudosas .................................... Reacciones negativas .................................. Total .............................................

5,964 3,415 104,266 113,645

(5.2%) (3.0%)

El resultado de estos exámenes indicaría que del 5 al 8% de los bovinos del Uruguay estarían infectados. LA INFECCIóN BRUCÉLICA EN OTRAS ESPECIES ANIMALES

La difusión de la brucelosis suina es poco conocida. Por los pocos datos existentes parecería que no estuviera muy extendida. La infección a Br. suis fué reconocida recién en el año 1943 (5), cuando se aisló por primera vez el germen en causa, pero ya anteriormente se habían encontrado aisladamente algunas muestras con reacciones dudosas y en 2 ocasiones con reacciones positivas. El primer foco de brucelosis suina, reconocido en el país, fué originado por la importación de una hembra de la República Argentina con reacción negativa, que abortó a los 3 meses en el establecimiento de destino, propagándose luego los abortos a 12 hembras más. De 95 suinos examina-

LA BRUCELOSIS ANIMAL EN EL URUGUAY

71

dos de ese establecimiento 42 resultaron positivos y 13 dudosos. Otros focos que se constataron a continuación fueron casi siempre originados por la importación de animales infectados, no reaccionantes a la seroaglutinación. El examen practicado por el Laboratorio de Biología Animal, en los años 1933-1941, en 204 muestras de suinos faenados en el Frigorífico Nacional de Montevideo, y 146 muestras de otras procedencias, dió por resultado 8 reacciones dudosas y las restantes negativas (5). En 1941, Scaltritti (3) examina 588 muestras de sangre de cerdos faenados en el Frigorífico Nacional, encontrando una muestra positiva. Pradines (3) practica la seroaglutinación en 751 muestras de suinos sacrificados en el mismo Frigorífico, encontrando sólo 4 reaccionantes dudosos. Según los datos que anteceden, parecería que la brucelosis suina está poco difundida en el Uruguay. La población caprina en el Uruguay es muy reducida. Según el censo de 1937 sería de 28,129 cabezas en toda la República. Las cabras no son explotadas en forma industrial, y el intercambio de especímenes es casi nulo. Esta circunstancia explica por qué esta especie animal no fué objeto de estudio en cuanto a infección brucélica. En los ovinos del país no se han encontrado reaccionantes positivos o dudosos en los 2,834 animales que se han examinado, procedentes de 8 departamentos de la República. El mismo resultado negativo se ha obtenido en cerca de 2,000 reproductores ovinos importados. LA LUCHA CONTRA LA BRUCELOSIS

Desde que la infección brucélica fué comprobada en el país, la Dirección de Ganadería se ha esforzado constantemente' por proveer al país de una legislación y de un plan de lucha para contener la difusión de la enfermedad y para reducir los estragos que ésta ocasiona. Los esfuerzos de la Dirección de Ganadería se han encontrado, sin embargo, con dificultades insalvables para lograr sus propósitos. La política profiláctica se puede dividir en 2 etapas, la primera, de 1928 a 1933, consistió en medidas severas y compulsivas para intentar erradicar la infección y la segunda, de 1933 a la fecha, tiene como base la profilaxis libre, opcional. Los progresos hechos, tanto en la primera etapa como en la actual, son muy poco satisfactorios, quedando como único saldo positivo la experiencia adquirida y la posibilidad de estructurar en el futuro un programa que se ajuste más a nuestra realidad epizootiológica y características de la explotación ganadera. Pasaremos rápidamente revista sobre la legislación profiláctica en el Uruguay, tratando de establecer en lo posible las circunstancias que se han opuesto a su ejecución. En 1928, en seguida de reconocida la infección, la brucelosis es incluida entre las enfermedades que dan lugar a la aplicación de las medidas sani-

72

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

tarias establecidas para las demás enfermedades infectocontagiosas (ley del 13 de abril de 1910). Esta inclusión, como asimismo el decreto de 21 de mayo de 1930 y del 16 de junio de 1932 implicaban la denuncia obligatoria de la enfermedad, el aislamiento de los animales infectados y la puesta del establecimiento bajo el control directo de la Dirección de Policía Sanitaria (hoy Dirección de Ganadería), estando ésta facultada para efectuar "las pruebas diagnósticas periódicas que crea necesarias". En la época en que fueron concebidas estas medidas, en el medio rural se sabia muy poco sobre la verdadera entidad y el alcance que tenía la infección brucélica para el ganado. La profesión veterinaria, por otra parte, desconocía el grado de difusión de la infección, por carecer de estudios nacionales sobre el tema, ni tenía la experiencia ni los medios necesarios para encarar la lucha contra la enfermedad. Estas medidas tan rigurosas se encontraron desde el principio con la resistencia de los ganaderos que veían lesionados sus intereses, y tuvieron que ser revocadas por el decreto de octubre de 1933. Con esa fecha "se faculta a la Dirección de Policía Sanitaria de los Animales para tomar en cada caso concreto en que se constata la infección, las medidas que la prudencia aconseje para localizar el mal y evitar su difusión". La Dirección de Ganadería de actualmente facilidades a los ganaderos que quieran sanear sus haciendas. A todo establecimiento en saneamiento se le ha eximido del pago por las serorreacciones que se efectúan en el Laboratorio de Biología Animal, siendo asimismo gratuito el servicio del veterinario que se traslada al establecimiento para extraer las muestras de sangre. La única exigencia es que el propietario "se comprometa a aceptar y dar cumplimiento a las indicaciones que se le formulen respecto al destino a darse a los sujetos reaccionantes". El único método oficialmente reconocido para el control de la brucelosis es el de la prueba serológica y la eliminación o aislamiento de los reaccionantes. La pérdida ocasionada por el sacrificio de los reaccionantes corre por exclusiva cuenta del propietario, no pagando el Estado ninguna indemnización. La vacunación con gérmenes vivos, tal como el empleo de la cepa 19, no está reglamentada y su elaboración, venta y uso son considerados ilícitos. Los resultados alcanzados por ese plan son muy poco satisfactorios. Un escaso número de establecimientos ganaderos se ha acogido a los beneficios otorgados. Por una parte, los conocimientos sobre la brucelosis y la manera de combatirla no tuvieron la difusión necesaria; por otra parte, es muy difícil conquistar la cooperación del hacendado para un programa de profilaxis opcional, exigiéndole sacrificios económicos. Si en Estados Unidos de Norte América el método de prueba serológica y sacrificio obtuvo algunos resultados significativos, es porque el gobierno federal y los estatales disponían de fondos necesarios para indemnizar a los propie-

LA BRUCELOSIS ANIMAL EN EL URUGUAY

73

tarios por sus pérdidas y pudieron de esa manera conseguir su colaboración. En nuestras condiciones, sin indemnización y sin un programa concreto de lucha con la correspondiente financiación para los gastos de operación, el procedimiento de prueba serológica y de sacrificio estaba desde el principio destinado al fracaso. En primer término, han recurrido lógicamente a la Dirección de Ganadería los hacendados que más tangiblemente han sufrido los estragos de la brucelosis, es decir, los propietarios de establecimientos donde la infección era de reciente instalación y de rápida difusión. En estos casos, como es sabido, el método de prueba y sacrificio es sumamente oneroso y casi siempre inoperante. Los propietarios de rodeos, con un índice bajo de infección, han sido los que en menor número se acogieron a las facilidades otorgadas, pero siempre que se actuó en estos establecimientos se obtuvieron resultados satisfactorios, con la diferencia de que la erradicación de la enfermedad era mucho más lenta en haciendas con un número elevado de animales, que en aquellas con un número más reducido. En términos generales, podemos decir afirmándonos en la experiencia adquirida, que no se puede construir, en el Uruguay, un plan de control de la brucelosis exclusivamente a base de prueba serológica y sacrificio. En el mejor de los casos, aun contando con la cooperación de los hacendados, no disponemos del personal técnico necesario para esa empresa. En efecto, en muchos departamentos, con una extensión de 8 a 10 mil kilómetros cuadrados, con varios cientos de miles de cabezas de ganado vacuno y muchos cientos de establecimientos ganaderos, hay un solo veterinario y que lógicamente, sólo en forma parcial, puede dedicarse a la lucha contra la brucelosis. No menos importante es el hecho de que por la característica de la explotación extensiva es sumamente difícil tomar las medidas de higiene indispensables, quedando muchas veces el material infeccioso por un tiempo indeterminado en el campo. En vista de los factores arriba mencionados, abogamos para que se incorpore en la lucha contra la brucelosis, mientras no haya un agente inmunizante mejor, la vacuna a cepa 19, porque entendemos que es la única manera de conquistar la cooperación de los hacendados y de extender la profilaxis de la brucelosis sobre todo el país. Desde el año 1942, la Dirección de Ganadería, por intermedio del Laboratorio de Biología Animal, está elaborando y aplicando la vacuna a cepa 19 en establecimientos ubicados en varios departamentos de la República, a título experimental y en forma gratuita. El Primer Congreso Nacional de la Brucelosis, realizado en Montevideo en diciembre de 1947, ha aprobado una moción presentada por los técnicos del Servicio de Brucelosis del Laboratorio de Biología Animal de la Dirección de Ganadería, solicitando a los Poderes Públicos que se autorice y reglamente la elaboración y uso de la vacuna a cepa 19.

74

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Esta iniciativa ha encontrado una acogida favorable en el ambiente ganadero, como lo atestigua la solicitud de la Asociación Rural del Uruguay dirigida al Ministerio de Ganadería y Agricultura, pidiendo se autorice el uso de la cepa 19, bajo el control estatal (6). El empleo de la vacuna a cepa 19 se encuentra actualmente a consideración de la Dirección de Ganadería. Entre las otras medidas preventivas, adoptadas por el Uruguay, mencionaremos el Reglamento de Importación y Exportación de Animales, del 8 de junio de 1934, y el Reglamento de Exposiciones, Ferias y Remates de Ganado, del 7 de octubre de 1942. En el primero de estos reglamentos se establece que todos los reproductores de las especies bovina, equina, porcina, caprina, y ovina, serán sometidos, mientras cumplen la cuarentena, a las pruebas diagnósticas para el reconocimiento de la brucelosis. De la importancia de esta medida hablan las cifras que arroja el suero-diagnóstico de animales importados entre los años 1943 y 1946. De 5,622 reproductores bovinos importados, de los cuales 5,437 corresponden a la República Argentina y los restantes de ultramar, 476 ó sea el 8.4% dieron reacciones positivas y 417 ó sea el 7.4% reacciones dudosas. El Reglamento de Exposiciones, Ferias y Remates establece que todo reproductor bovino o suino macho o hembra, de pedigree o puro por cruce, que concurra a exposiciones, ferias o liquidaciones de estancias, tiene que estar provisto de un certificado de "libre de brucelosis". Esta reglamentación tenía ante todo por objeto poner coto al fraude que significaba el vender reproductores infectados y de poco rendimiento por animales que, a juzgar por sus cualidades zootécnicas y de pedigree, serían de alto valor. En cuanto a la brucelosis suina, no hay ningún programa estatal de control, excepto los 2 reglamentos ya mencionados, debiéndose mencionar que la Dirección de Ganadería ha procedido en varias ocasiones, al constatar la infección, al sacrificio de todos los animales del establecimiento infectado, indemnizando al propietario. REFERENCIAS (1) Cassamagnaghi, A.: Enfermedad de Bang. Bol. Pol. Sanit. Anim., año 12, No. 1, p. 21, 1928. (2) Rubino, M. C.: El aborto epizoótico o enfermedad de Bang. Bol. Pol. Sanit. Anim., año 19, No. 10, p. 501, 1935. (3) Purriel, P., Risso, R., y Espasandín, J.: Brucelosis. Estudio de esta enfermedad en el Uruguay. Editorial Independencia. Montevideo, 1944. (4) Szyfres, B.; Rodríguez García, J. A.; Giacometti, H.; e Infantozzi, J. M.: Primer Congreso Nacional de Brucelosis. Memorias. Montevideo. p. 82, 1947. (5) Rubino, M. C.; Szyfres, B.; y Tortorella, A.: Núm. Cient. Acc. Sind., año 7, No. 1, p. 13, 1945. (6) Rev. Asoc. Rural del Uruguay, p. 56, sbre. 1949.

LA BRUCELOSIS ANIMAL EN EL URUGUAY

75

ANIMAL BRUCELLOSIS IN URUGUAY (Sunzmary) Bovine brucellosis was recognized in Uruguay in 1928. Of 113,645 bovine blood samples examined, 5.2% were positive and 3% suspicious. These samples came from 1161 ranges and farms, distributed all over the country. Of these herds 31.95% contain one or more reactors. Of the 224 dairies located in the capital, Montevideo, which were examined in 1932-33, 51.7% were infected. Of a total of 1172 of these animals 20.3% were positive reactors and 14.7% suspicious. All purebred and crossbred animals shipped to expositions or auction sales must be provided with a brucellosis-free certificate. The control-program in Uruguay consists of free assistance on behalf of the Bureau of Livestock to any herd owner who desires to eradicate the infection from his herd. Test and slaughter or test and segregation is the procedure employed. Only a few herds are under this program and up to the present, only a few have been freed from infection, especially the smaller ones and with a low percentage of infection. No indemnity payment and scarcity of personnel are the principal difficulties in this work. Strain 19 vaccination is now under the consideration of the Ministry of Agriculture and Livestock. Immunization with Strain 19 is being employed in a limited number of herds, under experimental conditions. Brucella suis infection in Uruguayan swine herds was recognized in 1943. It seems that swine brucellosis is not very widespread. Brucella infection in sheep and goats has not been found yet and Br. melitensis has not been isolated from other sources.

BRUCELLOSIS-A PACKING PLANT PROBLEM By NORMAN B. MCCULLOUGH, PH.D., M.D. MicrobiologicalInstitute of the National Institutes of Health, Public Health Service, Bethesda, Maryland A few years after the discovery of the etiology of Malta fever, the importance of raw goat's milk in the epidemiology of the disease in man was revealed. With the demonstration of brucellosis in man due to Brucella abortus and Brucella suis, further evidence accumulated of the importance of milk in the transmission of this disease. For some years raw milk was considered as the prime source of brucellosis in man. In the last decade or two with pasteurization of milk being practiced more extensively, it has become increasingly apparent that contact with infected animals and their tissues is now equally, if not more, important than milk in the spread of this disease to man. In the United States it would appear that the number of cases occurring after exposure to infected animals or their tissues may exceed the number deriving their disease from milk. The incidence in workers coming directly in contact with infected animals far exceeds that in the general population. In Iowa, in an investigation of 2,405 case reports of human brucellosis occurring over a seven year period, 75% of the cases gave a history of direct contact with farm animals.1 In a study of the epidemiology of 268 culturally proved cases in Minnesota, at least 60% were classified as occupational in origin.2 In Argentina, similar findings have been reported.3 Brucellosis is now recognized as an occupational disease. With the growing recognition of brucellosis as an occupational hazard, there has been much speculation and confusion in regard to the epidemiology and incidence of this disease in packing plant personnel. Opinions range from (a) that the exposure incidence is extremely low and the hazard relatively unimportant to (b) that simply walking through the packing plant or stock yard area constitutes exposure to brucellosis. The true incidence of human brucellosis in the general population is difficult to determine. If we were to count only those cases which are culturally proved, the number would be small. However, when we consider the high incidence of dermal sensitivity to Brucella and also of positive agglutination titer, it would appear that a large segment of the population has at some time come in contact with the organism. How many of these individuals may have had clinical undulant fever is unknown. Dermal sensitivity occurs in from 10 to 25% of the population in different parts of the country. In certain occupational groups, such as veterinarians and packing house workers, it is much greater than this. In tests of 11,000 sera derived from people living mostly in Chicago, some degree of agglutinins (1/25 or higher) was found in 1.55 per cent. 4 76

BRUCELLOSIS-A PACKING PLANT PROBLEM

77

In other areas where exposure to raw milk and infected animals is greater, the incidence is increased. In limited surveys of packing house workers, positive agglutination tests have been reported in from 10 to 30% of the workers.s, s Our knowledge of the extent of the hazard due to brucellosis in packing plants is incomplete. It is based largely upon the results of surveys employing dermal sensitivity and the agglutination test and a few well conducted epidemiological studies. These studies have not been extensive. Recently we have determined the presence of agglutinins in the personnel of three small specialty plants-a glue plant, a fertilizer plant, and an extract plant-located in the Chicago Stock Yards area. The standard test tube agglutination test was employed with incubation at 37 ° C for forty-eight hours, using our standardized antigen. The data obtained appears in condensed form in Table I. TABLE I.-Brucella agglutination titers of personnel employed in a glue plant, extract plant, and fertilizer plant Agglutination Titers Plant

Glue ........ Extract...... Fertilizer....

No. Men

294 77 133

~

0

146 24 57 7 102 16

45 9 12

27 3 3

16 1 0

12 0 0

7 0 0

4 0 0

6 0 0

2 0 0

~

No. Pos.

5 0 0

148150.34 2025.97 31 23.31

% Pos.

These plants were selected as representing end-points of the slaughterhouse processing line. The incidence of 50.34% positive tests obtained in the glue plant personnel speaks strongly for the intensity of exposure at this level and contrasts with the findings of 25.97% and 23.31% in employees working in the extract and fertilizer plants. The incidence of high titers was confined almost wholly to glue plant personnel. Of 148 glue plant workers with demonstrable agglutinins, 52, or 35.1%, had titers of 1/160 or higher. In the extract plant, only one of 77 men tested had a titer of 1/160, and in the 133 men working in the fertilizer plant there were no titers higher than 1/80. The marked difference in the reflected degrees of exposure is presumably due to the different nature of the materials handled in the respective plants. Commencing two years ago, we examined the submaxillary lymph nodes of slaughtered hogs by weekly sampling over a period of six months. 8S 9 One lymph node was excised from each carcass and cultured for Brucella. In this unselected series of 5,000 marketed hogs, Brucella was isolated in thirty-five instances, or 0.7% of the hogs. In addition to Br. suis, Br. abortus and Brucella melitensis were repeatedly recovered.

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Br. melitensis infection in hogs had been reported previously in Iowa. t° Since our preliminary report, Damon et al. have isolated this species from one hog in Indiana." There is growing epidemiological evidence that cases of Br. melitensis infection in man in this country are derived from hogs.2 11 '12 13 With the isolation of all three species of Brucella from hogs slaughtered in a large Chicago packing plant, it is apparent that persons engaged in the slaughtering of hogs and the processing of their meat are exposed to all three species. We have observed cases of undulant fever due to Br. abortus occurring in individuals working exclusively on the hog kill and have also reported a case of brucellosis due to Br. melitensis in a meat inspector engaged in inspecting the head glands of hogs.9 Our knowledge of the incidence of brucellosis in the hog is incomplete, but it appears high. Various surveys indicate that this incidence may be from 1 to 20%. 14' 15, 16, 1 In 1,008 hog sera o]btained during the previously mentioned survey, 40% were found to have agglutination titers of 1/20 or higher, and 6.45% of 1/160 or higher.9 If one were to take only the 0.7% of culturally proved brucellosis as the true incidence of infected carcasses giving exposure to the workers, in large plants, each individual handling the carcasses would come in contact with Brucella several times a day. If the incidence of high agglutination titers found in this survey is taken as the real incidence of infection, then "several" must be changed to "many". Since the inception of the brucellosis control program in cattle, great numbers of Bang's reactor cattle have been slaughtered. Workers handling these carcasses take no extra precautions. It has been assumed by some and all too often stated that these animals offered little risk to the worker. This opinion was based on statements that brucellosis in the cow is a localized disease and that only the uterus and udder are infectious. This, of course, is not so. Although there is this tendency to localize, there is ample evidence to show that the disease may be generalized for months after its inception. In a number of studies, Br. abortus has been recovered from the blood stream of cattle.' 8' 19, 20 Recently, Manthei and Carter studied both artificially and naturally infected cows. 2' Of 270 cows examined, positive blood cultures were obtained from 172. In eighteen animals studied for a prolonged period, 80% yielded positive cultures for five months, with bacteremia persisting in one animal for nearly two years. The organism has been isolated from many portions of slaughtered animals. In an attempt to obtain more direct data bearing on this problem, we recently cultured the carcasses of 100 Bang's reactor cattle slaughtered in a Chicago plant.22 Cultures were obtained from over twenty locations, mostly lymph nodes, throughout the carcass. As was to be expected, in a number of these cattle, no isolations were made. Br. abortus

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was recovered from some tissue in 42% of the animals. In 29%, isolations were made from locations other than the uterus and supramammary lymph nodes. In 10%, there were numerous recoveries from many locations. With the culturally proved incidence of brucellosis in slaughter-house hogs, the high incidence of agglutination titers found in these animals, and the high incidence of generalized infection in slaughtered Bang's reactor cattle, one must conclude that packing plant workers suffer relatively heavy exposure to Brucella from these sources. There has been much speculation concerning the role of air and other environs in the dissemination of brucellosis among plant personnel. Although such factors may contribute to the epidemiology, one would hardly expect them to be as major a factor as repeated daily contact with heavily infected animal tissues. Aside from the exposure offered by the fresh carcass, the viability of Brucella during processing of meat products may be important. It has long been known that the viability of Brucella is great. Recently, Hutchings et al. have studied the viability of Br. suis in the carcasses of slaughtered hogs subjected to the usual degree of refrigeration. 2 3 They were able to isolate Brucella from many locations in the carcass for as long as three weeks after slaughter. We have confirmed this fact and have recovered Br. suis from refrigerated lymph nodes forty-nine days after slaughter. In collaboration with Dr. L. M. Hutchings, we conducted experiments on the fate of Brucella in naturally infected pork when subjected to curing and smoking.2 4 A herd of infected hogs was obtained. When isolated, the infecting agent proved to be Br. melitensis. The hogs were slaughtered, and the hams and shoulders were subjected to the usual curing process employed in one of the large packing plants. Trained packing plant personnel carried out the procedures. Plant conditions were simulated as closely as possible. Cultures were made from the pickled hams and shoulders at various intervals. Br. melitensis was isolated repeatedly for as long as three weeks. A number of cured hams regularly yielding positive cultures were then subjected to smoking. This procedure was carried out under packing plant conditions. During smoking the hams are subjected to relatively high temperatures. No isolations were made after smoking. It would appear that during the smoking process Brucella is destroyed, but that the organism survives during pickling. Extensive and comprehensive surveys of the personnel of packing plants to determine how many of the workers actually contract brucellosis have not been made. Such studies, employing not only the usual serological and dermal sensitivity techniques, but also blood cultures, history taking, physical examinations, and competent appraisal of the health status of each worker examined, would do much to clarify our

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knowledge of the incidence and extent of clinical brucellosis in this group of workers. The packing industry has been reluctant to accept brucellosis as an occupational disease. However, for a number of years our South American neighbors have recognized brucellosis as an occupational and compensable hazard. Three years ago the state of Iowa similarly recognized its importance and passed an occupational disease law to cover the situation in that state. This enlightened attitude is to be commended. As Borts has pointed out, "That brucellosis is an occupational hazard is not denied by those who are fully informed and are willing to admit facts and potentialities as they exist." 25 Until such time as brucellosis in domestic animals is eradicated or so reduced in incidence as to be negligible, the occupational hazard of brucellosis will continue. The question arises as to what precautions might be instituted to reduce the danger. At present in most plants none are taken. It is difficult to formulate a plan until all the facts are at hand. Here we are still badly in need of facts. Further investigations of the incidence and epidemiology of the disease are needed. The people working in these plants should be studied to delineate the degree and extent of disability involved. There are some protective measures that could be used even now. An educational program seems indicated. Individuals should be urged to report any illness or injury promptly. Even minute cuts and abrasions should be given prophylactic care. Personal hygiene should be stressed. It would be quite feasible to go even further than this, particularly in the case of Bang's reactor cattle. Such material might even be slaughtered separately by workers already immune to the disease. Education would appear to be the prime tool in this program. REFERENCES (1) Jordan, C. F.: Symposium on Brucellosis, A. A. A. S. The Epidemiology of Brucellosis, pp. 98-115, 1950. (2) Magoffin, R. L.; Kabler, P., Spink, W. W.; and Fleming, D.: An epidemiologic study of brucellosis in Minnesota. Pub. Hlth. Rep., 64: 1021-1043 Aug. 19, 1949. (3) Molinelli, E. A., Basso, G., and Miyara, S.: Epidemiologia de la brucelosis humana en la República Argentina. Rev. Asoc. Med. Arg., 61: 182-188, 1947. (4) Unpublished data. (5) Huddleson, I. F.: Brucellosis in man and animals. New York, The Commonwealth Fund, 1943. (6) Topley and Wilson's Principles of Bacteriology and Immunity, 3rd ed., Baltimore, The Williams &Wilkins Co., 1946. (7) McCullough, N. B.: Symposium on brucellosis, A. A. A. S., Laboratory Tests in Brucellosis, pp. 116-121, 1950. (8) McCullough, N. B.; Eisele, C. W.; and Pavelchek, E.: Isolation of Brucella abortus from hogs. Pub. Hlth. Rep., 64: 537-538 Apr. 29, 1949.

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(9) McCullough, N. B.; Eisele, C. W.; and Pavelchek, E.: A Survey of brucellosis in slaughtered hogs. Pub. Hlth. Rep. In press. (10) Borts, I. H.; McNutt, S. H.; and Jordan, C. F.: Brucella melitensis isolated from swine tissues in Iowa. Jour. Am. Med. Assoc. 130: 966, 1946. (11) Damon, S. R., and Scruggs, J. H.: Recovery of Brucella melitensis from the hog. Pub. Hlth. Rep., 65: 374 Mar. 17, 1950. (12) Jordan, C. F., and Borts, I. H.: Occurrence of Brucella melitensis in Iowa. Jour. Am. Med. Assoc. 1S0: 72-75 Jan. 12, 1946. (13) Kabler, P.; Bauer, H.; and Nelson, C. B.: Human Brucella melitensis infections in Minnesota with hogs as the probable source. Jour. Lab. & Clin. Med., 32: 854-856, 1947. (14) Boak, R. A., and Carpenter, C. M.: Brucella abortus agglutinins in porcine blood. Jour. Inf. Dis., 52: 425-429, 1930. (15) McNutt, S. H.: Incidence and importance of Brucella infection of swine in packing-houses. Jour. Am. Vet. Med. Assoc., 39: 183-191, 1935. (16) Boyd, W. L.; Kernkamp, H. C. H.; Roepke, M. H.; and Blye, C. E.: Incidence of brucellosis in swine. Proc. 46th Ann. Meeting U. S. Livestock San. Assoc., pp. 124-128, 1942. (17) Hutchings, L. M.: Brucellosis in swine. Proc. 47th Ann. Meeting U. S. Livestock San. Assoc., pp. 52-58, 1943. (18) Fitch, C. P.; Bishop, L. M.; and Kelly, M. D.: The isolation of Brucella abortus from the blood stream of cattle. Proc. Soc. Exp. Biol. & Med., 34: 696-698, 1936. (19) Lubke, A.: tUber das Vorkommen von Bang-bakterien im Blut von Kihen aus banginfizierten Bestanden, Ztschr. f. Infektionskr., 47: 240, 1935. (20) Soule, M. K.: Bacteriological and serological findings in Brucella abortus infections in animals and man. Premier Congrés International de Microbiologie, 1: 606, 1930. (21) Manthei, C. A., and Carter, R. W.: Persistence of Brucella abortus infection in cattle. Am. Jour. Vet. Res., 11: 173-180, 1950. (22) McCullough, N. B.; Eisele, C. W.; and Byrne, A. F.: Incidence and distribution of Brucella abortus in slaughtered Bang's reactor cattle. Pub. Health Rep. In press. (23) Hutchings, L. M. et al.: In press. (24) In preparation for publication. (25) Borts, I. H.: Some observations regarding the epidemiology, spread and diagnosis of brucellosis. Jour. Kansas Med. Soc., 41: 399, Dec. 1945. LA BRUCELOSIS COMO PROBLEMA DE LAS PLANTAS DE ENVASE (Sumario) En los Estados Unidos el número de casos de brucelosis humana observados después de la exposición a animales infectados o los tejidos de éstos, parece exceder el número de los que contraen la enfermedad por la leche. En una investigación de 2,405 casos de brucelosis humana observados en Iowa, 75% acusaron antecedentes de contacto directo con animales de granjas. En un estudio epidemiológico de 268 casos comprobados por medio de cultivos en Minnesota, por lo menos 60% se clasificaron como ocupacionales. Se han comunicado hallazgos semejantes en Argentina. La brucelosis se reconoce ya como enfermedad ocupacional. La frecuencia de la sensibilidad dérmica a la brucela y de las aglutininas del

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suero es mucho mayor en grupos ocupacionales específicos que entre la población general. Se examinó en busca de aglutininas en el suero al personal de tres pequeñas plantas de envase: una planta de cola, una planta de abonos, y una planta de extractos, que representan los puntos terminales de las operaciones de un matadero. En el personal de la planta de cola se encontró que la frecuencia de las reacciones positivas ascendía a 50.34%, llegando a 25.97 y 23.31%, respectivamente, entre los empleados de la planta de extractos y de abonos. Los títulos elevados se limitaron casi por completo al personal de la planta de cola. En una serie no seleccionada de 5,000 cerdos mercados, se aislaron brucelas de los nódulos linfáticos submaxilares de 35 (0.7%). Se descubrieron las tres especies de Brucellas, encontrándose por primera vez la Br. abortus en los cerdos infectados naturalmente. Se ofrecen pruebas epidemiológicas indicativas de que el cerdo constituye una fuente de las infecciones humanas por Br. abortus y Br. melitensis. Se examinó la carne de 100 cabezas de ganado que habían acusado reacciones positivas para la Br. abortus. Se prepararon cultivos con 23 tejidos, principalmente nódulos linfáticos. Se aisló la Br. abortus de una o más localizaciones en 42 animales. En 10 de los animales se aisló de numerosos sitios. Aunque el útero y los nódulos linfáticos supramamarios fueron los órganos invadidos con más frecuencia, se encontró la infección en zonas muy esparcidas. Los perniles y cuartos delanteros de los cerdos infectados naturalmente con Br. melitensis fueron sometidos al proceso comercial de salazón. La Br. melitensis se aisló repetidamente de los jamones en conserva durante tres semanas. No se aisló la brucela después del ahumado. Se discute el riesgo ocupacional de la brucelosis en la industria de envases.

VARIATION AS A TOOL IN BRUCELLOSIS RESEARCH

By WERNER BRAUN, ARTHUR N. GORELICK, MARY KRAFT AND DOROTHY D. MEAD Camp Detrick, Frederick, Maryland The striking differences in colonial types of Brucella are usually associated with differences in virulence. They, therefore, provide simple markers for determining the fate of variants in vivo after simultaneous or consecutive inoculation of two or more variant types. It is of considerable value for various phases of brucellosis research to utilize such marked cell types for in vivo studies. First, by adjusting infective doses of consecutively administered inocula in such a manner that ID 50 's are comparable, it becomes possible to study certain immunologic and pathogenetic problems. Since descendants of the first inoculum can be easily distinguished from the progeny of the second inoculum by observing colony-types on plates streaked with tissue samples from the infected animals, the proportion of cells recoverable from the primary or secondary infection can be ascertained rapidly in any tissue. Second, this technique permits the investigation of certain problems associated with a chronic disease such as brucellosis. Previous in vitro studies had shown that the less virulent nonsmooth types do not establish themselves in originally smooth cultures which are maintained in the presence of small amounts of normal serum from susceptible animals. In contrast, a rapid establishment of these less virulent nonsmooth variants was observed in vitro in the presence of sera from infected or immunized animals. These effects were shown to be independent of the presence of agglutinins. On the basis of these results it was believed that less virulent nonsmooth types may also establish themselves in vivo some time after an acute infection but may be replaced eventually by virulent types when selective conditions return to the status analogous to normal hosts. It was suggested that such population changes may play a role in the frequently observed relapses. The findings in one extensive experiment with guinea pigs, in which an intermediate variant of Br. suis gradually replaced a smooth type, seemed to substantiate this assumption. However, it was not possible to detect a similar replacement of smooth types by nonsmooth types in several subsequent guinea pig experiments. In order to obtain further information on this point, as well as information of the kind which we discussed earlier, guinea pigs were inoculated subcutaneously or intracardially with massive doses of a mucoid variant of Brucella suis one week or five weeks after primary infection with smooth organisms. The intervals of one week and five weeks between S and M infections were chosen because in vitro studies had shown that the selective activity of guinea pig sera was unaltered one week after S 83

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infection but had undergone changes five weeks following S infection. The smooth Brucella suis cells were injected subcutaneously in the right inguinal region; each animal was infected with 30 cells which represents approximately 10 ID60 's. One group of animals was then inoculated one week later with 2 X 109 mucoid cells per animal subcutaneously near the site of the primary infection; another group was inoculated intracardially one week after the smooth infection. Two additional groups of animals were similarly inoculated 5 weeks after the primary S infection. A total of 80 guinea pigs were thus infected; in addition 21 control animals were infected with S only, 24 other animals were infected subcutaneously or intracardially with M cells only. Animals were sacrificed 1, 2, 4, and 6 weeks following the inoculation of M cells and 17 different tissues of each animal were cultured after thorough grinding with mortar and pestle. The distribution of recoverable M cells differs significantly depending on the route of inoculation. One week after the subcutaneous M injection, M cells were recoverable only from the right inguinal lymph node, left axillary lymph node, spleen, liver, renal or lumbar lymph node, and to some extent from the bone marrow. In contrast, all tissues from intracardially challenged animals sacrificed at the same time permitted the recovery of a generally high percentage of M type cells. The relatively low percentage of M type cells recoverable from the right inguinal lymph node and the lumbar lymph node of these "intracardial" animals is noteworthy, especially since these two tissues yielded the highest recovery of M types in subcutaneously challenged animals. This may be attributable to some kind of blocking effect towards the intracardially introduced M cells in the tissues closest to the site of the primary infection. In all instances the lumbar or renal lymph node yielded recoveries identical to those of the regional right inguinal lymph node, just as if there were a direct connection between those two lymph nodes. In any event a comparison between the two groups of animals indicates that a blocking effect by the lymphatic system follows the subcutaneous administration, whereas intracardial infection permits a general distribution of the variant type throughout the body of the host. The next point of interest is the rapid disappearance of the M cells from most tissues within 4 weeks after infection. The different tissues seem to possess a differential ability to rid themselves of the M types, as shown by the greater persistence in the mesenteric and cervical lymph nodes. It was also observed that the M type disappears much faster from the tissues of animals previously infected with S type cells than from previously uninfected animals. Practically no M cells were isolated from animals sacrificed 2 weeks or more after the secondary M infection, whereas in M infected, previously unexposed, animals the

same M type was found to persist for more than 4 weeks.

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The disappearance of M types is even more rapid in animals which were challenged with M 5 weeks after S infection. No M types could be recovered at all from the tissues listed one week after subcutaneous M infection of previously S-infected animals. M types could actually be recovered from all these animals when abscesses near the site of infection were cultured. Whereas intracardial M challenge one week after S infection permitted good recovery of M types for at least two weeks, a similar challenge 5 weeks after the S infection yielded no M recoveries two weeks later. In addition to these recoveries from various tissues, localized abscesses found in a number of animals were cultured. Most of these abscesses yielded M cells even when they came from animals which did not permit recovery of M cells from any of the other tissues. It was possible to isolate M cells from an abscess of an animal (No. 30) four weeks after the subcutaneous M infection. At that time no M cells could be isolated from any other tissue. An animal (No. 60) which was sacrificed 6 weeks after intracardial M infection contained an abscess in the submaxillary lymph node which yielded 5% M and 95% S cells. The following results resembled the Koch phenomenon: When M cells were injected subcutaneously into animals 5 weeks after the S infection, no M cells could be recovered from any of the usual tissues cultured, but for 2 weeks following the M infection enormous numbers of M cells were found in localized abscesses at the site of injection. Two more examples showed the survival of less virulent nonsmooth types in localized lesions four and six weeks after intracardial M infection or 9 and 11 weeks after the S infection. One animal had a lung abscess containing 100% M cells, the other animal had a pericardial abscess containing 98% nonsmooth cells of a rough type plus 2% S cells. (Incidentally, cervical and bronchial lymph nodes, as well as the spleen, appear to be preferential sites for long persistence of S cells.) In reviewing these data we must conclude that, at least as far as the nonsmooth type used in these studies is concerned, there appears to be no opportunity for the less virulent type to establish itself for prolonged periods in what we might call the "open" tissue of the host, even though it was introduced at a time when the selective serum activity of the host had changed. However, less virulent nonsmooth types, either introduced by injection or appearing spontaneously in vivo, seem to be able to persist in localized abscesses. Further studies are required to confirm these isolated observations, but it should be pointed out that such behavior would be in agreement with recent results obtained in test tube experiments. Studies reported by Goodlow, Mika, and Braun revealed that population changes in vitro are controlled by the accumulation of a toxic metabolite in originally smooth cultures. It was demonstrated that Brucella cells produce alanine as an end-product of normal me-

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tabolism. When alanine accumulates to a certain level in the culture it inhibits the growth of smooth types but permits the establishment of spontaneously arising nonsmooth mutants with greater alanine-resistance. These nonsmooth types continue to produce alanine and will be replaced eventually by other, even more alanine-resistant, nonsmooth types, or in some strains, by a highly alanine-resistant smooth type. Preliminary evidence also indicated that the addition of serum from a normal, susceptible host may modify the metabolism so that no alanine is produced and population changes are avoided. While this information was of great value for the understanding and control of variational processes, it also suggested that selective phenomena may be entirely different in vitro and in vivo. The data cited had shown that the accumulation of a specific metabolite plays a major role in the creation of specific environmental conditions which will favor the nonsmooth variants, but this accumulation took place in test tubes, i.e. in a closed system. The animal body probably does not represent such a closed system but rather an open system from which accumulating metabolites are removed continuously. Therefore, it would not be expected that conditions which will affect population changes in vitro will be created to the same extent in most tissues of a host. However, a high metabolite concentration could be expected in a closed environment as represented by abscesses. Localized abscesses may, however, constitute the site at which the nonsmooth types find the environmental conditions that are necessary to endow them with a high selective value. The value and significance of the previously reported serum effect, i.e. the activity of the SS factor in vitro, does not appear to be diminished in view of these results. Time does not permit citation of unreported data which further illustrate how closely the in vitro activity of the SS factor correlates with stages of immunity and susceptibility, but we may add that it is entirely possible that the establishment of less virulent types in vivo may require both the availability of a closed system and an alteration of the SS factor activity of the host. The obvious question whether it might be possible to modify the course of the disease by raising the in vivo level of metabolites involved in favoring the establishment of less virulent types cannot be answered as yet, but it illustrates the new type of approach to economically important problems which can be developed by using variation as a tool in brucellosis research. LA VARIACIóN COMO UN INSTRUMENTO EN LA INVESTIGACION DE LA BRUCELOSIS (Sumario) Las diferencias en los tipos de colonias, que por lo común indican diferencias de la virulencia, facilitan puntos de referencia sencillos para comprobar el destino de las variantes in vivo después de la inoculación simultánea o consecutiva de dos o más tipos variantes. Para comprobar la posibilidad de que las variantes no

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lisas menos virulentas de la Brucella suis persistieran in vivo después de las infecciones prolongadas con microorganismos lisos sumamente virulentos (S), se inocularon cobayos con dosis masivas de una variante mucoidea (M) una semana o cinco semanas después de la infección primaria S, utilizando la via subcutánea o intracardiaca. Los animales fueron sacrificados a la semana, dos semanas, cuatro semanas y seis semanas después de la inoculación de las células M, cultivando una gran variedad de los tejidos. La distribución y persistencia de los tipos M en los distintos tejidos variaron en forma notable según la via de inoculación. Después de la administración subcutánea se observó bloqueo del sistema linfático. Los diversos tejidos revelaron al parecer distinta capacidad para desembarazarse del tipo M. Con excepción de algunos abscesos localizados, los tipos M no persistieron en ningún tejido durante períodos prolongados. Se presentan los resultados de estos experimentos, así como la relación que guardan con los resultados de los estudios in vitro, y se discuten los problemas de la patogenia.

THE BACTERICIDAL ACTION OF HUMAN BLOOD AGAINST BRUCELLA AND ITS SPECIFIC INHIBITION* By WENDELL H. HALL, M.D., PH.D. Laboratory Service of the Veterans Administration Hospital and the Department of Medicine, University of Minnesota Medical School As a part of a study of the pathogenesis of human brucellosis an investigation of various serologic changes was carried out. A review of the literature indicated that, whereas such antibodies as agglutinins and opsonins have been studied thoroughly, little attention has been paid to bactericidal antibodies in human brucellosis. Considerable work has been carried out with the blood of various animals (principally cows). Only the most pertinent observations will be reviewed here. In 1932 Rundberg' published a thesis concerning the bactericidal action of human and animal sera upon Br. abortus in which he concluded that the bactericidal antibody did not increase upon immunization. He

found the antibody was heat-stable, but also decided that it did not require the presence of complement in order to lyse Brucella. The latter observation was contrary to the findings of Mackie and Finkelstein 2 and many subsequent investigators. 3 4, 6 Irwin and Ferguson 6 found that upon recovery from Br. abortus infection the titer$ f bactericidal antibody in the serum of cows increased from 1:25 or less to 1:625 or more. More recently Huddleson 7 noted that the plasma bactericidal activity of Brucella infected cows was less than that of normal cows when tested against Br. abortus. He did not reconcile this observation * Sponsored by the Veterans Administration and published with the approval of the Chief Medical Director. The statements and conclusions published by the author are the result of his own study and do not necessarily reflect the opinion or policy of the Veterans Administration.

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with the work of Irwin and Ferguson. 6 In 1947 the present author 8 reported similar observations with the blood of normal humans and patients having brucellosis. It was pointed out at that time that the reduction in bactericidal activity of the serum of patients infected with Brucella was due to the prozone inhibition phenomenon. The actual titer of the bactericidal antibody had increased during the disease. This observation has subsequently been confirmed in animal experiments by Berman and Irwin 9 and by Huddleson.'° It is the purpose of the present communication to offer an explanation for the mechanism of this prozone of inhibition of bactericidal activity. The methods employed in the studies to be reported here have been given in detail in a thesis submitted to the faculty of the Graduate School of the University of Minnesota and will be published elsewhere. They will therefore be described only briefly here. (1) The "standard" bactericidal test. Serial tenfold dilutions of a suspension of Brucella were added to equal quantities of fresh defibrinated blood, serum or plasma. After 24 hours incubation at 37°C a loopful of each mixture was subcultured upon an agar plate in order to demonstrate the viability of the Brucella. The bacterial inoculum was controlled in each experiment by four plate colony counts. (2) The "dilution" bactericidal test. The "standard" test was altered through the use of serial tenfold dilutions of serum inactivated at 56°C for 30 minutes. After the addition of Brucella, complement was added. Since guinea pig complement was ineffective, either diluted fresh infant's serum or rabbit serum was used as the source of complement. (3) Absorption of Brucella antibodies was carried out with bacterial antigens prepared by the addition of diethyl ether to heavy suspensions of the organisms in physiologic saline solution. After evaporation of the ether, nine volumes of the antigen were added to one volume of the normal or immune serum. After incubation for 2 hours at 37°C and 18 hours at 5°C the mixtures were centrifuged and the supernatant fluid was tested for antibody content and re-absorbed with the same antigens. Serial absorptions were carried out 3 to 4 times in most cases. Observations in normal human subjects.-The blood of well over 100 children and adults who did not have brucellosis was examined for Brucella antibodies including bactericidins. The age of the subjects appeared to influence the presence or absence of bactericidins significantly. Although the seruff of infants 3 months or less in age was strongly bactericidal for Br. abortus, two thirds of the infants between 4 and 16 months of age had little or no bactericidal activity. The serum of all children 18 months of age and older, as well as that of all normal adults, was strongly bactericidal for Br. abortus. The antibody was undoubtedly transferred passively to young infants from their mothers. The pathogenesis of its appearance in older children remains a mystery comparable to that which surrounds the development of iso-hemagglutinins.

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The velocity of the lethal effect of human serum upon Br. abortus was moderately rapid; the majority of bacterial cells were killed within 2 to 4 hours at 370°. The remaining bacteria, however, were killed only at a much slower rate, suggesting that two factors were operating, a fact which was soon confirmed. While fresh normal adult serum was strongly bactericidal for Br. abortus, this property was almost completely lost after heating to 56°C for 30 minutes. However, the addition of diluted fresh serum from an infant or rabbit, which was not bactericidal per se, restored the adult serum to its full activity. Thus, it became apparent that the bactericidal effect of serum depended upon the presence of a heat-stable substance (antibody) and a heat-labile substance (complement). Another factor which governed the bactericidal phenomenon was the variety of Brucella. It was conclusively demonstrated that, whereas Br. abortus was easily killed by normal human serum, Br. suis and Br. melitensis were resistant to its action. This observation was of interest in view of the greater virulence of the latter varieties for man. It was also noted that the temperature of incubation had a marked influence upon the ability of human serum to kill Br. abortus. Both serum and citrated whole blood from normal adults and patients having acute brucellosis, though actively bactericidal for Br. abortus at 37°C, had little lethal effect upon these bacteria at 40C. In fact, we have isolated viable organisms after a small inoculum (5 cells per ml.) of Br. abortus was introduced into citrated blood and stored 125 days at 4°C. This fact is of obvious importance in the operation of blood banks. All donors should be carefully screened in order to rule out brucellosis if transmission by transfusion is to be avoided. The inhibitory effect of low temperatures upon this bactericidal system appeared to be due to the fact that complement failed to unite with the antibody-sensitized bacteria. The principal factors responsible for the bactericidal property of human blood for Br. abortus appeared to reside in the serum or plasma and not in the cells. The latter, by themselves, had little or no such activity. However, whole citrated or defibrinated blood from normal adults and from patients having brucellosis was often more strongly bactericidal for Brucella than plasma or serum. The difference was not well correlated with the opsonic index of the blood, and it may have been due to nonopsonic phagocytosis of the Brucella. Dilution of normal adult serum beyond a certain point removed its bactericidal activity even though the complement was restored. It was established that the maximum titer of the heat-stable bactericidal antibody for Br. abortus ranged from 1:42 to 1:420 in the serum of 6 normal adults. A fact of considerable significance was disclosed by absorbing normal human sera with various bacterial antigens. It was found that the heatstable bactericidal antibody for Br. abortus was absorbed equally well by

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Br. abortus, Br. melitensis and Salmonella oranienburg. Since the latter organism contained no antigen in common with Brucella, it must be concluded that the natural bactericidal antibody is not specific. Observations in patients having brucellosis.-No qualitative difference was disclosed between normal human bactericidal antibody and that found in the serum of patients suffering from brucellosis. It soon became apparent however that, although the serum of patients having acute brucellosis (symptoms for less than 3 months) was strongly bactericidal for Br. abortus, the undiluted serum of patients who had the disease for a long period of time was not bactericidal. These statements are based upon the study of the blood of 119 patients who were judged to have brucellosis. The diagnosis was proved by the isolation of Br. abortus in 38, Br. suis in 1 and Br. melitensis in 3 patients. The most unequivocal data on this point were gathered by analysis of 79 bactericidal tests using the serum of 22 patients having proved brucellosis. Br. abortus was isolated from 20 and Br. melitensis from 2 of the patients. None had prior Brucella vaccine therapy nor skin tests. In those patients whose symptoms had been present for one month or less, the mean bactericidal power of the serum for Br. abortus was high (10 - 6 dilution of Brucella sterilized). At the end of 2 months this figure had declined to 10- 7 , and by the end of 3 months it had reached 10-8. The mean serum bactericidal power did not rise above this figure thereafter though the symptoms of the disease had been present for 2 years or longer. The serum of none of the patients killed a significant number of Br. suis or Br. melitensis at any stage of their disease irrespective of the variety of infecting Brucella. The importance of obtaining serum before Brucella vaccine therapy or skin tests was emphasized by the observation that the ability of the undiluted serum of patients to kill Br. abortus sharply declined shortly after the intradermal injection of several Brucella antigens. Cholera vaccine also caused a similar decline in the bactericidal activity of human serum for Br. abortus, as well as an increase in serum Brucella agglutinins. This was attributed to the fact that Brucella contain an antigen similar to the H antigen of Cholera vibrio. Though the (undiluted) serum of patients having prolonged symptoms of brucellosis often failed to kill Br. abortus, whole defibrinated or citrated blood from the same patients was often strongly bactericidal. The difference in activity was not accounted for by specific opsonins; it was not well correlated with the opsonic index (Huddleson). The blood cells from such patients were not bactericidal per se. The presence of "nonspecific" opsonins might explain the difference. The low bactericidal activity of the (undiluted) serum from patients having chronic brucellosis was regularly increased during therapy with such antibiotics as streptomycin, aureomycin and chloramphenicol. Streptomycin therapy enabled the blood to kill large numbers of Brucella

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of all varieties. Aureomycin had somewhat less effect, and chloramphenicol had the least. The effect of dilution of the serum upon its bactericidal power for Br. abortus was studied by a method allowing restoration of complement activity. When tested by this method, the titer of heat-stable bactericidal antibody was 1:42,000 in 1 patient and 1:420,000 in 2 other patients having acute brucellosis. These patients had been ill for 2 months or less. Repetition of such experiments with serum from patients having chronic brucellosis was of greater interest. While such sera were usually unable to kill Br. abortus before dilution, after dilution to 1:420 or even 1:42,000 they became strongly bactericidal. One such serum manifested bactericidal activity even after dilution to 1:4,200,000. It was clearly evident that the lack of bactericidal activity on the part of such (undiluted) sera was due to the prozone inhibition phenomenon. The mean titer of maximum bactericidal activity against Br. abortus was 1:4,200 in the serum of 15 patients having brucellosis and manifesting such prozones. The average duration of symptoms in these patients was 13 months (range 2 months to 20 years). When non-bactericidal (undiluted) sera from patients having brucellosis were added to an equal quantity of normal human serum, the resulting mixture was not bactericidal for Br. abortus. In other words, prozonal sera contained an inhibitor preventing bactericidal action against Br. abortus by normal bactericidal antibody. There was no evidence that complement activity was interfered with. The titer of this inhibitor was determined in the serum of 15 patients having chronic brucellosis. It ranged from 1:7 to 1:42,000 (mean = 1:420). The titer of the inhibitor in all sera was always less than the titer of maximum bactericidal activity. The inhibitor was heat-stable and was not inactivated in the presence of crystalline human or bovine albumin. Brucella antibodies were absorbed from the serum of several patients having acute brucellosis due to Br. abortus. Bactericidins for Br. abortus were completely removed by one absorption with either Br. abortus, Br. melitensis or S. oranienburg. It was concluded that the bactericidal antibody found in the serum of patients having acute brucellosis was non-specific and did not differ from natural bactericidal antibody. In a similar fashion Brucella antibodies were absorbed from the serum of several patients having chronic brucellosis by repeated exposure to bacterial antigens. After 1, 2 and occasionally even after 3 absorptions with Br. abortus or Br. melitensis these non-bactericidal sera became increasingly bactericidal for Br. abortus though their Brucella agglutinins and opsonins had been completely absorbed. After 4 absorptions the sera lost their bactericidal antibody. The phenomenon was not due to serum dilution. Serial absorption of these sera with S. oranienburg

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did not remove Brucella agglutinins nor opsonins nor did it cause the sera to become bactericidal for Br. abortus. Addition of absorbed and unabsorbed sera from patients having chronic brucellosis to normal human serum indicated that absorption with Br. abortus and Br. melitensis antigens removed the bactericidal antibody inhibitor. Absorption with S. oranienburg did not do so. The bactericidal antibody inhibitor in such sera therefore appeared to be specific for Brucella. Confirmation of this was given by the fact that (undiluted) serum from patients having chronic brucellosis, though not bactericidal for Br. abortus, was strongly bactericidal for S. oranienburg. The facts appear to justify the conclusion that the inhibition prozone, which appeared in the serum of patients having brucellosis for several months and rendered their (undiluted) serum non-bactericidal for Br. abortus, was not due to the production of an excess of (non-specific) bactericidal antibody but was the consequence of the stimulation of a specific inhibitor which prevented the lethal activity of that antibody (plus complement) upon Br. abortus. Sera containing this inhibitor became bactericidal for Brucella only after absorption with Brucella or after the addition of antibiotics capable of killing Brucella. No blood product was found which was capable of overcoming the inhibitor. REFERENCES (1) Rundberg, G.: Eine Experimentelle Studie iber die Immunitatsverhaltnisse bei Bang's Abortus Bazillus. Thesis, Svenska lak-sallsk. forhandl. 58: 77-152, 1932. (2) Mackie, T. J., and Finkelstein, M. H.: Natural bactericidal antibodies; Observations on the bactericidal mechanisms of normal serum. Jour. Hyg. 31: 35, 1931. (3) Irwin, M. R., Beach, B. A.; and Bell, F. N.: Studies on the bactericidal action of bovine whole blood and serum towards Brucella abortus and Brucella suis. Jour. Inf. Dis. 58: 15-22, 1936. (4) Shrigley, E. W., and Irwin, M. R.: On the differences in activity of serum complement from various animal species. Jour. Immunol. 32: 281-290, 1937. (5) Irwin, M. R., and Beach, B.A.: Differential bactericidal activity of bovine serum toward strains of Brucella abortus of high and low virulence. Jour. Agr. Res. 72 (No. 2): 83-91 Jan. 15, 1946. (6) Irwin, M. R., and Ferguson, L. C.: Increase of bactericidins in the serum of cattle following recovery from infection with Brucella abortus. Proc. Soc. Exp. Biol. and Med. 38: 451-452, 1938. (7) Huddleson, I. F., Wood, E. E., Cressman, A. R. and Bennett, G. R.: The bactericidal action of bovine blood for Brucella and its possible significance. Jour. Bact. 50: 261-277, Sept. 1945. (8) Hall, W. H. and Spink, W. W.: Studies of immunity in brucellosis: The bactericidal action of human blood against Brucella, Proc. Am. Soc. Clin. Invest., Atlantic City, May 1947. Abstract, Jour. Clin. Invest. 26: 1183, Nov. 1947. (9) Berman, D. T., and Irwin, M. R.: Further studies of the bactericidal action of

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bovine serum for Brucella. Proc. 48th Meeting Soc. Am. Bact., Minneapolis, May, 1948, pp. 38-39. (10) Huddleson, I. F.: The potentiating action of sulfonamides on the Brucella antibody-complement system. Am. Jour. Vet. Res. 9: 277-285, 1948.

ACCIÓN BACTERICIDA DE LA SANGRE HUMANA CONTRA LA BRUCELA Y SU INHIBICIÓN ESPECIFfCA (Sumario) Desde hace cerca de veinte años se ha reconocido que la sangre del hombre y animales normales es capaz de destruir las brucelas, especialmente la Brucella abortus. Los experimentos realizados por el autor han demostrado que los anticuerpos bactericidas humanos son termoestables, pero que requieren la presencia de un complemento (de hombre o de conejo) para la máxima actividad. Asi-

mismo se demostró que eran activos en la ausencia de leucocitos, que eran activamente bactericidas a 37° C pero no a 4° C, y que eran absorbidos tan ávidamente

por la Salmonella oranienburg como por la brucela, y que por lo tanto eran noespecíficos. El titulo de anticuerpos bactericidas en sueros de personas normales varía entre 1:42 y 1:420. El suero de enfermos de brucelosis aguda era fuertemente bactericida para la Br. abortus. El titulo de los anticuerpos bactericidas de tales sueros variaba entre 1:42,000 y 1:420,000. Los anticuerpos bactericidas inmunes no se diferenciaban cualitativamente de los anticuerpos bactericidas normales, y eran también noespecíficos. El suero no diluido de la mayor parte de los enfermos, que han padecido de brucelosis durante dos o más meses, no pudo destruir la Br. abortus en número apreciable. Esta falta de actividad bactericida se debe al fenómeno de inhibición de prozona. Dichos sueros resultaron bactericidas para la Br. abortus al diluirse de 1:420 a 1:42,000 y activarse con complemento. Las mezclas de dichos sueros con suero normal no resultaron bactericidas a menos que se diluyeran mucho los primeros. El suero no bactericida, sin diluir, contenía un anticuerpo inhibidor bactericida. Este inhibidor fué la causa del fenómeno de prozona, y su titulo era menor que el de los anticuerpos bactericidas. No obstante, dicho inhibidor era especifico para la brucela y se combina a está con mayor avidez que los cuerpos bactericidas. La actividad bactericida de dicho inmunisuero podía aumentarse

con la adición de antibióticos como estreptomicina, aureomicina, y cloranfenicol.

CLINICAL COURSE OF HUMAN BRUCELLOSIS IN MINNESOTA By WESLEY W. SPINK, M.D. AND ROBERT L. MAGOFFIN, M.D. Department of Medicine, University of Minnesota Hospitals and Medical School Studies on global epidemiology in recent years have emphasized that the clinical manifestations of an infectious disease may vary from one geographical area to another. Brucellosis is a disease that reflects a variable clinical course, even when studied in one locality. A classification of the signs, symptoms and duration of brucellosis based upon observations in a limited geographical area may provide misleading information when applied to other regions. For example, the disease varies according to the species of Brucella that is prevalent in a given area. The over all clinical picture of the disease due to Brucella melitensis differs from that caused by Brucella abortus. There is also a variation in the animal reservoir of the disease throughout the world, embracing such factors as the species of animal harboring the infection, and the number of animals infected. Local habits and customs will greatly influence the opportunity of human exposure to the disease. The underlying state of health of a regional population will have an important bearing on the severity and duration of the illness. In view of these variables, it is desirable to have reliable data on the natural course of the human disease in a given area to serve as a sound basis for evaluating therapeutic measures. For these reasons, it is essential to carry out detailed studies of human brucellosis as it exists in different localities. As a contribution to this effort, this report is based upon personal observations made on a group of patients in Minnesota during the years of 1937 to 1950. ECOLOGY OF BRUCELLOSIS IN MINNESOTA

Minnesota possesses a large rural population engaged in dairy farming and the raising of hogs. There is an extensive reservoir of brucellosis in the dairy herds, approximately 20% of the herds showing one or more reactors for Bang's disease. There are several large meat packing plants in the state, and thousands of animals are brought in from neighboring states for processing. In general, the people are an economically independent, hardy pioneer stock of Nordic ancestry and in an excellent state of nutrition. Animal and human brucellosis in Minnesota is primarily a disease due to Br. abortus, though hogs in this area serve as a reservoir for both Brucella suis and Br. melitensis. Several factors have permitted a comprehensive study of the human 94

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disease as it exists in this state. The population is relatively stable. The referring physicians have been most cooperative, and the administrators in the packing plants have been very helpful in our investigations. It should be emphasized that the University Hospital is a state institution, receiving patients predominantly from small towns and rural areas. Since Minnesota embraces a recognized endemic area of brucellosis, the hospital staff has been alerted for many years to the presence of the disease. Adjacent to the University Hospitals are the Laboratories of the Minnesota State Department of Health with well trained personnel and available diagnostic techniques. Diagnostic services are not only available without charge to physicians by the Minnesota Health Department but the Department has also carried on a consistent campaign for years urging physicians to utilize these services, particularly cultural techniques. As a result, physicians throughout the state have been on the alert for the disease and have channelled both questionable and problem cases of brucellosis into the hospital. It is significant that although several hundred patients have been examined for brucellosis each year, the diagnosis of active disease has been established in only 186 patients between 1937 and 1950. This number is in contrast to the larger numbers of cases reported by other investigators in the United States. This discrepancy reflects the difference in criteria utilized for the diagnosis of active brucellosis, a feature which will be discussed shortly. COMPOSITION OF CLINICAL MATERIAL

Of the 186 cases, 101 were proved bacteriologically (Table 1). Only 18 were females and the remaining 168 were males. This preponderance of males over females is due in part to the inclusion of a large number of employees of packing plants among the cases. The over all sex distribution for all reported cases in the State of Minnesota is 78.5% males and 21.5% females. The age distribution of the University Hospital cases is detailed in Table 2. The preponderance of cases was in males in the age group of 20 to 45 years, which reflects the high proportion of cases having an occupational source of infection among farmers and packing plant employees. While the University Hospital serves primarily the rural population in the state, only 30% of the cases originated in rural areas. The large number of urban cases is due again to the preponderance of packing plant employees. The occupation and residence of all the cases are summarized in Table 3. It should be stressed that the proportion of cases with severe illness and complications was greater than usually encountered, because the severity of the disease prompted the physicians to seek hospitalization for the patients in this category.

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TABLE 1.-Cases of brucellosis observed. University of Minnesota Hospitals and

Clinic, 1937-1950 Total cases diagnosed ........................................... Brucella isolated ............... ........ .. .............. Br. abortus.............................................. 89 Br. suis .......... ........................... 7 Br. melitensis ........................................... 5 TABLIE

186 101

2.-Age and sex distribution. 186 cases of brucellosis Males

Females

0-4 5-9 10-19

2 2 9

0 O 2

20-29

58

3

30-39

61

4

40-49 50-59

21

2 5

23l

60-69

3

2

5

Totals

168

18

186

Age Group

12

Both Sexes

Per Cent

8.1

61}

67.7 21.5

2.7

TABLE 3.-Occupation and residence. 182 cases of brucellosis Rural

Farmers and farm laborers....

Urban

44

Farm wives ....................

8

Farm children .................

2

Packing plant and stockyard workers ....................... Laboratory personnel ........... Veterinarians .................. Housewives and children ........ Miscellaneous trades ............

Total ......................

54

Total .........................

DEFINITION OF "ACUTE"

AND "CHRONIC"

88 6 2 12 20 128

BRUCELLOSIS

Because of the variability in the manifestations of brucellosis, it is difficult to define the disease in terms of "acute" brucellosis and "chronic" brucellosis. Since these terms have been used with different and conflicting interpretations by various workers, it is not easy to employ them without some misunderstanding. Therefore, we should like to make it clear that we are using the terms "acute" and "chronic" primarily with reference to the duration of the illness. Thus "acute" brucellosis signifies an illness of short duration. In addition, according

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to common usage, it also implies a degree of severity. A "chronic" illness means one of long duration, and as a corollary, an illness usually not manifested by severe symptoms. But brucellosis may be manifested by a brief and mild illness, and a long, protracted illness may have severe and debilitating complications. In this report, however, the terms "acute" and "chronic" will apply principally to the duration of the illness, regardless of the severity. As in previous statements, we will regard brucellosis as being acute, only if the illness has endured for 3 months or less. We have previously considered a case chronic, if the illness extended beyond 3 months. However, in the light of an extended follow-up study of the disease, we now reserve the term "chronic" for those cases in which illness endured for at least 1 year or longer. This is more in accordance with the frequent use of the term "chronic brucellosis" in the literature to designate an illness of several years' duration. For an illness of intermediate duration of 3 to 12 months, we will employ the term "subacute." MANIFESTATIONS OF EARLY STAGES OF BRUCELLOSIS

In order to formulate as accurately as possible the clinical manifestations and course of the disease as it occurs naturally with little or no alteration by effective therapy, we have made a careful analysis of 68 patients observed for the most part before the advent of the antibiotics. None of these patients received aureomycin, chloramphenicol or the combination of streptomycin and sulfadiazine. The majority of these patients did receive one of the sulfonamides at some time during their illness and six patients received a short course of streptomycin. A few patients also received vaccine. About one-fourth of this group received no chemotherapy or vaccine. Though it is possible that the illness in some of these patients was favorably influenced by the therapy received, it is now generally recognized that the sulfonamides and streptomycin, when used alone, do not consistently alter the pattern of disease. These 68 patients have been included in the "untreated" group. Brucella were isolated from 44 of the 68 patients. The species of Brucella included 40 strains of Br. abortus, 2 of Br. suis, and 2 of Br. melitensis. Three of the patients died within the first year of the disease of subacute bacterial endocarditis due to Br. abortus, leaving 65 cases for a long follow-up study. All of the patients have been followed for a minimum of one year and the majority for over three years, while some have been seen intermittently for 7 to 13 years. The average follow-up period has been 4.5 years. It should be emphasized again that many of the patients included in this study were seriously ill, and there were several individuals with painful and disabling complications. The incubation period of brucellosis in these patients, when it could be determined, was found to vary from a few days to several months.

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The onset varied widely. Some patients experienced a gradual onset with a sense of general malaise and a mild indisposition to prolonged physical and mental activity. Fever was detected upon examination, but often the patients did not realize that they had an elevated temperature. On the other hand, in many of these patients after a prodromal period of a day or two, the onset was very abrupt and severe with the appearance of chills, fever, sweats and headache. The most constant symptoms elicited were weakness and a tendency to fatigue more readily than usual. Other common manifestations were headache, a feeling of chilliness or the presence of rigors, nocturnal sweating, generalized aches and nervousness. The most constant objective physical finding was the presence of fever. A tendency to "nervousness" associated with tremors of the hands was frequently observed. The palms and soles were usually moist and mottled in appearance. A majority of the patients demonstrated enlarged cervical lymph nodes, and less commonly, axillary and epitrochlear lymphadenopathy. Splenomegaly was demonstrable in about one-third of all the cases, but usually was associated with the more severely ill patients. The laboratory findings during this early phase of the illness were unequivocal. In 44 patients the organism was isolated. Agglutinins were demonstrable in all cases in a titer of 1:80 or higher, though in one or two instances the initial test was negative because the patients were seen so very early in their illness. The most reliable laboratory procedure for screening the suspected cases of brucellosis was the agglutination test, utilizing the tube-dilution method with an antigen of Br. abortus obtained from the Bureau of Animal Industry of the United States Department of Agriculture. Misleading information has found its way into the literature on the agglutination reaction in brucellosis which has been based on the use of unreliable antigens, and the employment of rapid slide techniques of doubtful accuracy. One unsubstantiated and highly misleading statement repeated over and over is that "organisms are frequently isolated from patients who have a negative agglutination reaction." All too frequently the studies cited to support this assertion make no mention of the type of agglutination test employed or the antigen used. Convincing evidence supporting such a statement in a significant number of cases in which reliable techniques were used is lacking. But there is considerable available evidence that if a reliable antigen is used with the tube-dilution technique, one rarely will isolate Brucella from patients with absent agglutinins, and only infrequently from patients having a low titer of agglutinins. The Laboratories of the Minnesota Department of Health routinely culture approximately 800 specimens of citrated whole blood submitted annually from suspected cases of brucellosis (1). During the four year period of 1945-1949, approximately 3200 samples were cultured, from which 350

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isolations of Brucella were made. Only two of the bloods from which Brucella were cultured failed to show the presence of agglutinins. All the other bloods from which the organisms were isolated revealed a titer of 1 to 80 or above. None had a maximum titer of only 1 to 40.. It is significant that the majority of the 3200 bloods studied had either absent agglutinins, or a low titer of agglutinins, so that ample opportunity was afforded for the discovery of organisms from patients of this type. Similar evidence has been accumulated by Damon (2) in Indiana where apparently a less sensitive antigen is employed for the agglutination test. Clots from all bloods submitted by physicians for Brucella agglutination test were cultured. In a study of 7906 clotted specimens, 23 isolations were made from the 513 specimens which revealed agglutinins in a titer of 1:40 or above. Of the remaining 7393 clots which showed no agglutinins present in a titer of 1 to 40 or above, only two isolations were made. In our experience, extensive cultural studies have been made of the blood, bone marrow, liver biopsies, lymph nodes and spleen, and no isolations of Brucella have been accomplished except in those patients having agglutinins. When a correlation is made between the titer of agglutinins and positive blood culture, over 90% of our patients have had a titer of 1 to 160 or above. Since many normal and healthy individuals in Minnesota have a low titer of Brucella agglutinins, a titer of less than 1:160 frequently has doubtful clinical significance (3). It has been our practice to obtain at least three specimens of blood for culture on three different days in suspected cases of brucellosis. Brucella have been cultured from approximately 50% of our cases of brucellosis. In three instances, organisms have been recovered from aspirated sternal bone marrow, when simultaneous cultures of venous blood have remained sterile. Other helpful aids in screening the early case of brucellosis have been the total leukocyte and differential blood counts. Usually the total count has been normal or reduced, and has rarely exceeded 10,000 cells per cu mm. A relative lymphocitosis was usually present. It is of interest that since individuals entering the University Hospitals come from an endemic area of brucellosis, approximately 20% of all adult patients were found to exhibit a positive reaction following the intradermal injection of Brucella antigen. Neither the intradermal test nor the opsonocytophagic reaction has been found to be a reliable diagnostic aid, and these tests are not used by our group. The clinical and laboratory data which have been described to this point constitute the characteristic features of the initial or early phase of illness as seen in our patients. In a number of cases the early phase had completely or almost completely subsided before the patient came under our care.

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This initial period of the illness which is referred to by some authors as the acute phase, represents the period of relatively sustained symptoms until a temporary remission or recovery ensues. In the 65 untreated cases previously referred to, the initial attack lasted from three to four weeks up to eight or nine months. In a substantial majority, however, the early phase of the illness lasted from one to three months. SUBSEQUENT COURSE OF BRUCELLOSIS

Little controversy exists over the clinical manifestations which have been described for the early phase of brucellosis, but general agreement is lacking as to the total duration of the illness and ultimate outcome of the patients. In attempting to determine when the patient had fully recovered from the disease, we have depended upon objective and subjective data. A patient was not considered as being restored to normal health unless he said so. He was still considered as being ill if he complained of symptoms, even though no objective findings were demonstrable. Acute brucellosis.-Of the 65 living patients, 10 or 16%, recovered completely within three months, and have remained well for one to seven years. Subacute brucellosis.-There were 25 cases in which the illness endured for three to twelve months. This group comprised 39% of the cases. The disease persisted in these cases because of first, the initial phase frequently associated with persistent bacteremia, endured beyond three months; second, the patients experienced one or more relapses after periods of relative well-being lasting for several weeks or months; and third, there was a protracted period of convalescence without objective evidence of active disease being present. A demonstrable complication was responsible for prolongation of the illness in only one of the patients, and this one patient had encephalo-meningitis. Thus, thirty-five of the total number of patients, or 54% having subacute or acute illness had recovered completely within one year (Table 4). Chronic brucellosis.-This group included 30 cases or 46% of the total, who were ill for one year or longer. These patients can be divide into one of three groups. First, were those individuals having evidence of continued active infection, which was manifested by recurring relapses, but without evidence of localization. Both laboratory and clinical evidence of active disease was present, such as was found in the initial phase of the illness. There were only five cases in this group. All of these five patients eventually recovered, and have remained well for one to three years. The second group of cases with an illness lasting for more than a year included 12 individuals who had localizing evidence of the disease associated in some cases with frequent recurrent relapses of systematic

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illness. The localized complications observed in these 12 patients were: five of spondylitis; two of meningo-encephalitis; one of meningo-encephalitis and spondylitis; one of cholecystitis; one of pericholecystic abscess; one of bone lesion of right hip; and one of radiculo-neuritis. Not included in this group of complications are the three patients who died of subacute bacterial endocarditis. The third group of chronic cases comprised 13 individuals in whom all objective evidence of active illness had subsided, but who still complained of ill health. No significant evidence of disability was present. These 13 cases represented one-half of the patients who had been ill for more than a year, or 24% of all the cases. It is this group of patients that represents the difficult problem of chronic brucellosis today. Fever TABLE 4.-Duration of symptoms in 65 cases untreated brucellosis. (1937-1950) 37 Br. abortus, 2 Br. suis, and 2 Br. melitensi No. Cases

Per cent

Acute ....................

10

15

Subacute ................ Chronic .................

25 30

39 46

was not demonstrated in any of these patients after 12 to 18 months of illness. Repeated cultures of blood remained sterile, and the agglutinin titer had gradually diminished. It should be emphasized that in all the cases of chronic brucellosis it is most unusual in our experience to isolate Brucella from cultures of blood after the illness has endured for one year. Because these 14 patients presented a difficult therapeutic problem they have been studied very thoroughly, and several features of their illness stand out. In the first place, several of these patients had underlying emotional difficulties. Two of the patients had been discharged from the armed services during World War II because of inability to adapt prior to the onset of brucellosis. Two others were alcoholics of long standing. Only three of the patients were females, and each of them had had difficulty in meeting their daily problems. One cannot deny that this group of patients were ill. It is well known that brucellosis does have a serious impact upon the nervous system, and that underlying personality disorders and conflicts may be intensified by the disease, but after fever and other objective evidence of active infection have subsided, it is our view that nothing is to be gained by continuing to direct therapy against presumptive infection that cannot be demonstrated. On the contrary, much harm may be done by encouraging the patient to feel that he had a chronic illness for which nothing can be done and, therefore, he need not make any further attempt to cope with the exigencies of earning a living. He is sick; he is blameless for his failures. Another

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factor that has entered into the prolonged illness of some of these patients, has been financial compensation for disability under the Workmen's Compensation Act. Brucellosis is a compensable disease in the meat packing plants of Minnesota. A careful study of some of these patients has convinced us and our associates that the convalescence has been prolonged because of compensation received or because of unsettled claims for compensation. And finally, there is a very small group of chronically ill patients who appear to have been emotionally stable individuals prior to illness, yet who have had protracted symptoms. In the absence of objective evidence of localization of the disease of continued active infection, it is our conclusion that this type of illness again represents a post-infectious syndrome, which is more directly attributable to a reaction pattern set up during the course of the active disease. TABLE 5.-Duration of disability in 65 cases untreated brucellosis Months

No. Cases

Per cent

0-3 3-6 6-9 9-12 12-24

23 14 13 7 8

35 22 20 11 12

Of the 30 chronically ill patients, 18 have been observed to recover completely, and they have remained well for one to seven years. Six of these 18 patients had an illness persisting for two to five years. The remainder of these 18 patients were recovered by the end of two years. In 12 of these 30 chronic cases, the patients still had complaints at the time of the last follow-up so that the ultimate duration of illness could not be determined. Thus, in our series of 65 patients, over 80% are known to have recovered completely and have remained well for at least a year when last seen, and 31 cases are known to have remained well for three or more years. In our experience then, the great majority of patients have had a self limiting illness with subsequent recovery. PERIOD OF DISABILITY CAUSED BY BRUCELLOSIS

In the foregoing discussion, we have been concerned with the total duration of time in which the patients had symptoms. An analysis of the total period of disability has also been made. A patient was considered disabled if he could not carry on with the routine daily work as he could before he became ill. This included those individuals who were only partially disabled. There is a marked contrast between the duration of illness when based on subjective complaints, as compared

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to disability caused by the disease (Table 5). While 30 of the 65 cases, or 46%, complained of symptoms beyond one year, only 8 cases, or 12%, were disabled beyond that period. In fact, 37 of the cases, or 57%, were able to return to their usual routine at the end of 6 months, and 77% at the end of nine months. Factors prolonging disability were a relapsing illness and the presence of complications. In addition, there were those individuals who could not endure the day's work, although no organic cause for their disability could be ascertained. REFERENCES (1) Bauer, H.: Personal communication. (2) Damon, S. R. and Albright, K.: The isolation of Brucella from blood clots. Brucellosis. Am. Assoc. Adv. Sc., p. 122, Washington, D. C., 1950. (3) Spink, W. W., and Anderson, D.: Brucella studies on bank blood in a general hospital. Jour. Lab. and Clin. Med., 35: 440, 1950. LA EVOLUCION CLÍNICA DE LA BRUCELOSIS HUMANA EN MINNESOTA (Sumario) El estudio clínico de 186 casos de brucelosis humana observados en Minnesota durante un periodo de 13 años, ha recalcado el hecho de que la enfermedad se limita a si misma. La clasificación de la brucelosis clínica propuesta se basa en la duración de la enfermedad desde la iniciación de los síntomas. Los casos en que la enfermedad ha durado menos de tres meses se clasifican como agudos, de tres meses hasta un año como subagudos, y cuando la enfermedad persiste durante más de un año se clasifica como brucelosis crónica. En Minnesota la inmensa mayoría de los casos de brucelosis se deben a la Br. abortus. En un grupo de 65 enfermos que no recibieron los medicamentos considerados eficaces en la brucelosis, se observó otro rasgo notable en la evolución clínica. El grupo comprendió 24 (39%), cuyos síntomas persistieron durante un año después de la iniciación de la enfermedad, pero sólo 8 (13%), no pudieron reanudar sus labores diarias al cabo de un año. El problema básico de la brucelosis crónica afecta hoy día como a 20% de todos los casos; este es un grupo de enfermos relativamente pequeño, pero importante. Estos son los sujetos con síntomas subjetivos, pero que revelan pocos signos clínicos convincentes o pruebas de laboratorio indicativas de enfermedad activa.

THE NATURAL COURSE OF BOVINE BRUCELLOSIS* By D. T. BERMAN Department of Veterinary Science, Wisconsin Agricultural Experiment Station, University of Wisconsin Bovine brucellosis (Bang's disease, contagious abortion) is an infectious disease, due in the majority of cases to Brucella abortus, and characterized by inflammatory changes of the endometrium and placenta, and often resulting in the premature expulsion of the fetus. The dramatic nature of the cardinal sign of abortion, and the lack of other manifest signs of disease has led to the development of a concept of bovine brucellosis as a placental disease, ignoring the often presented evidence for its generalized nature. All too often efforts at control are based upon this concept rather than upon the complete picture of the infection (see Elberg and Silverman).' In this paper an attempt will be made to describe the events in the natural course of bovine brucellosis, emphasizing the generalized nature of the infection, as demonstrated by many investigators. Because of time limitation the discussion of certain aspects of the disease, which do not bear directly upon this central theme and which have been reviewed adequately by others, will be curtailed. ROUTES OF INVASION

The earliest demonstrations of the infectious nature of bovine contagious abortion were accomplished by reproducing the typical syndromel2 3' 4 with intra-vaginal inoculations of pregnant cows with portions of fetal membranes, or uterine exudate discharged in abortions. Bang 4 with Stribolt in 1897 proved the etiological significance of the organism now known as Brucella abortus by producing typical abortions after intra-vaginal instillation of pure cultures isolated from the uterine exudate of an aborting cow. In this work epizootological evidence incriminating the bull as a source of infection for cows was also presented. Although it was shown' that bulls may develop genital infection, with shedding of organisms in the semen, after instillation of cultures into the prepuce or after intravenous inoculation, 6 the evidence for transmission to cows by natural service by such bulls has been inconclusive. 7 More recently Bendixen8 and Seit 9 have reported the spread of infection to previously clean herds by the use of artificial insemination of semen from infected bulls. Experimental confirmation has been obtained by Manthei et al.0 Agglutinins in seminal plasma have been described by Christensen."l * Published with the approval of the director of the station. 104

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While it is not improbable that contamination of the vagina with infected bedding, or by the use of infected semen, may be responsible for naturally acquired infection, it appears that ingestion is the most important means by which the organisms gain access to the body. Bang' 2 and others have shown that it is possible to reproduce the disease in pregnant heifers and cows by feeding infectious material or cultures, and it has been suggested that infection probably occurs through contaminated feed. The large numbers of organisms present in the uterine discharges of infected animals, and their resistance to most natural conditions with the exception of direct sunlight,' 3 14 offer sufficient opportunity for the contamination of pastures and feed. Experimentally it has been found possible to infect cattle through the abraded, or even intact skin.?5 Infections may also be produced by instillation of infective material into the conjunctival sac.l 6 The latter method has been employed to a great extent in research on bovine brucellosis because of its simulation of natural infection through a mucous membrane, and because it is possible to administer known numbers of organisms to all experimental animals. Of interest for further investigation are the reports of infection by arthropod vectors,' 7 by intrammary inoculation' 8 and by the respiratory route.l1 INFECTIVE NUMBER OF ORGANISMS FOR NORMAL CATTLE

The results of a number of trials with conjunctival exposure of normal cattle with varying numbers of either of two strains of virulent Br. abortus have been reviewed recently.2 o 21 In the series with one strain (528), exposure to approximately 15 million organisms produced results which were not significantly different from those obtained when the dose was 150 million or 1 billion organisms (55 abortions in 61 animalswith an infection rate somewhat higher). When the dosage was reduced to approximately 1 million organisms a significant reduction in the abortion rate was obtained (8 abortions in 19 animals) while the reduction in infection rate was not so great (13 infected of 19 animals). Less than a million organisms caused 3 abortions in 9 animals, with 5 animals infected, and somewhat more than a thousand organisms caused no abortions in 9 individuals, 2 of which became infected. The titration of the second strain (2308) showed no difference in abortion or infection rates in animals given 26 or 15 million organisms (29 abortions in 30 animals, all of which were infected). With 740 thousand organisms 7 of 9 animals aborted, and 8 were infected. A dose of 370 thousand produced abortions in 5 of 9 animals, and infections in 7 of these. From these results it would appear that there is a real difference in the virulence for cattle of these two strains of Br. abortus, and this

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impression is confirmed by the results of titrations in guinea pigs. 2 ' Further trials using a variety of strains, and larger numbers of animals will be necessary to arrive at numbers for general application. In investigational work the number of organisms used must be conditioned by the questions which the animals are being used to answer. SUSCEPTIBILITY OF NORMAL CATTLE TO INFECTION

A summary20 of the results of exposing normal, pregnant animals by the conjunctiva to at least a million organisms (of a variety of strains)usually many times this number-showed that in 36 trials there had been 303 abortions among a total of 353 animals. Analysis of the results indicated that the variations of the percentages of abortions around the average (85.8%) would occur infrequently by chance alone, meaning that the underlying probability of abortion could not be considered uniform from trial to trial. Among the variables encountered were differences in the stage of pregnancy at which animals were challenged, and the number of organisms of many different strains used. Sexually mature, unbred heifers have been considered relatively resistant to infection by Br. abortus. This opinion is supported in part by the small percentage of reactors in this classification observed in official testing programs, and in part by the study of Edgington and Donham, 22 in which 15 heifers exposed before breeding failed to abort during the subsequent pregnancy, and apparently failed to become infected. This apparent resistance, manifested particularly by failure to abort, has been the goal of prophylactic vaccination with living cultures. While the resistance engendered may protect against abortion, the vaccinated animals may become infected with the vaccine strain, and may shed the organisms in the milk, and/or uterus. Definitive evidence of the susceptibility of unbred animals has come from a number of workers, and here it is necessary to differentiate between the infectious disease, characterized by abortions, and infection per se. Bang et al.23 produced infection in 8 unbred heifers by feeding cultures. The infection was manifested by the dissemination of the organisms through the blood stream to various body lymph nodes of all, the liver of one, the spleens of 4, and the udders of 4. The nongravid uteri were not invaded. Unfortunately agglutination test results are not given in this paper. Manthei 21 has also reported an outbreak of brucellosis in a group of open heifers naturally exposed. Six of 24 animals developed agglutinins, Brucella were isolated from the udder secretions of 5, and from the uterine discharges of one of the 3 animals which had normal calves after the subsequent breeding. Approximately 3 years after exposure organisms were recovered from the tissues of 5 of the animals. Manthei also stated that a number of non-pregnant heifers have been experimentally exposed with resultant bacteremia and udder infection. Similar results4 have been obtained in our laboratory.

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The resistance of sexually immature animals is apparently of a different order. Calves infected in utero or from their surroundings after birth remain infected for a short time only unless they are kept in contact with infectious material, or fed infected milk. Within several weeks from the time calves are removed from the presence of the organisms they are usually free from infection, and develop into uninfected, susceptible cattle. While Carpenter 2 5 has shown that organisms fed young calves invade the lymph nodes of the head and digestive tract, and in a few cases may be found in the spleen, or liver, Brucella are eliminated within 4 to 5 weeks after the cessation of feeding. An attempt 2 6 to overcome the resistance of young animals by the administration of estrogenic hormones during exposure was unsuccessful. Further investigations of this type using more animals, and various hormone preparations would seem desirable. DISTRIBUTION OF BRUCELLA IN THE BODY OF THE HOST

A review of the studies of the distribution of the organism in the host emphasizes the significance of extra-genital sites of infection. Schroeder and Cotton2 7 tested by guinea pig inoculation the tissue of a group of naturally infected cows for the presence of Br. abortus. The tissues examined included the blood, spleen, liver, kidney, brain, ovary, uterus, milk, synovial fluid, and lymph nodes from all parts of the body. With the exception of the udders and supramammary lymph nodes, the organism was isolated in only one instance-from a pelvic lymph node. Birch and Gilman28 recovered Br. abortus from the supramammary lymph nodes of 7 of 17 cows, the lymph nodes of the head of 2 of 19, the bronchial and mediastinal glands of one animal, and the spleen of one. The mammary gland and inguinal lymph nodes were found to be infected in each of 3 cows examined by Roots and Ridala.29 The spleens of 2 animals yielded Br. abortus, as did the fluid of a carpal hygroma. Krüger30 isolated Br. abortus from the muscular pillars of the diaphragm of 2 animals which failed to react to the agglutination test. One of the most definitive studies was that of Bang, Bendixen and Oerskov2 3 who studied the distribution of Br. abortus in the bodies of 8 non-pregnant heifers fed large numbers of organisms, and killed at intervals from sixth to the ninetieth day after exposure. They found that in early stages of infection the organism was located principally in the lymph nodes of the head and digestive tract. After a month the bacteria were distributed throughout the reticulo-endothelial system. In animals killed later the organisms were found primarily in the mammary glands and supramammary lymph nodes. Doyle3 ' made a detailed examination for the presence of Brucella in the tissues of 32 naturally infected cows, and made recoveries from one or more sites in 26 animnals. Of the 13 tissues from which isolations

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were made 8 were lymph nodes, and Doyle concluded that the principal site of Br. abortus in the carrier cow, in addition to the udder, appears to be the lymphoid tissue. In an unpublished study3 2 Br. abortus was isolated from the supramammary lymph nodes of 13 of 33 reactor cows eliminated from herds in the test and slaughter control program. Isolations from various lymph nodes including those of the head and thorax, pelvis, and digestive tract have been made from 5 animals examined in this laboratory. The livers of 3 of these animals and the spleens of 2 were also infected. Manthei and Carter33 have tabulated the results of a study of the sites of localization among 15 experimentally infected cows, on which complete hemocultures were also performed. Isolations were made only from the reproductive tract, mammary gland and various lymph nodes. Infection of the supramammary lymph nodes was always associated with mammary infection. Brucella infection of the blood stream of infected cattle was demonstrated by Soule 34 and Fitch et al., 36 and a comprehensive study has been made recently by Manthei and Carter.3 3 The latter authors concluded from their results that the incidence and persistence of bacteremia may be used as criteria for the estimation of degrees of resistance and susceptibility of cows exposed to virulent Brucella. Bacteremia was not demonstrable in vaccinated animals which resisted the induced infection, while in unvaccinated susceptible animals there was a high incidence and persistence of blood stream infection. Strain 19 vaccinated animals not fully protected but with varying degrees of resistance showed a transient bacteremia. Persistent bacteremia was more frequently associated with abortions and weak calves at parturition than with normal calvings. Bacteremia persisted for 5 months in a high proportion of experimentally infected unvaccinated cattle, after which the incidence declined. Approximately 5 to 10% of the animals continued to exhibit intermittent bacteremia for 2 years. Manthei and Carter 33 have reviewed the studies of uterine infection in cattle. Of particular interest is the work of Fitch et al.34 in which Br. abortus was isolated from 24 of 66 uteri showing no evidence of recent parturition, and from 23 of 27 where there were signs of recent parturition. The organism was found in one non-gravid uterus 195 days after parturition. Similarly Birch and Gilman 28 have reported finding Br. abortus in the genital mucosa of 4 artificially infected animals 15, 13, 8 and 7 months respectively following parturition. These authors considered that true localization had occurred. In the study of Fitch et al.36 41 gravid uteri were examined, and Br. abortus was not isolated from any in which pregnancy of less than

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four and a half months existed. The organism was isolated from 45% of those more than four and a half months in gestation. Gwatkin 3 7 and Fitch et al.38 isolated Brucella from the uterine material of approximately 20% of reactors at the time of full term parturitions. From these studies it is possible to reconstruct a more complete picture of the course of natural infection in cattle. Following invasion through mucous membranes the organisms set up infection in the regional lymph nodes, multiplying therein for a variable period of time. They are then carried by the lymph to the blood, from which they may be isolated regularly or intermittently depending upon the numbers and frequency with which they are showered from the foci of infection. Dissemination to other lymph nodes, liver and spleen, the mammary gland and uterus is probably hematogenous. Invasion of the gravid uterus rarely occurs before the second trimester of pregnancy, and persistent localization following an infected parturition is probably relatively infrequent. In most of the 20% of animals which become persistent uterine shedders the reproductive tract is probably reinvaded during subsequent pregnancies, while in some animals true localization in the uterus apparently occurs. Among the gaps in this picture is the part played by the bone marrow as a site for localization. PATHOLOGY OF BOVINE BRUCELLOSIS

Considering the emphasis which has been placed on uterine infection, it is not surprising that most studies of the pathology of bovine brucellosis have been confined to the changes found in the uterus and mammary gland. Hallman's 3 9 study of the necrotic and inflammatory changes in the uterus and those of Ridala4 0 and Runnels and Huddleson 4l on udder inflammation have been reviewed adequately by Huddleson. 4 2 Histopathological examination 4 3 of infected lymph nodes reveals granulomatous changes similar to those described in the lymph nodes of infected swine by Brown et al.44 and in human bone marrow by Sundberg and Spink.45 The lesions observed in infected lymph nodes consisted primarily of a reticular hypertrophy and hyperplasia, with almost complete replacement of the medullary cords and parts of the cortex by swollen reticulum cells. There was also a lymphoid hyperplasia. Early focal changes were infiltration with polymorphonuclear leucocytes, monocytes and many plasma cells and proliferation of the capillary endothelial cells. Occasional giant cells could be seen, but necrosis was not apparent. In older lesions the lymphoid tissue was replaced by stellate collections of reticulum cells with pyknotic nuclei, which seemed to represent an effort at healing in the absence of fibrosis. These were analogous to the "reticulum cell scars" described by Brown et al.44

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The major route of elimination of the organism from the body of the infected cow is with the discharges from the uterus at parturition. Fitch et al.4 6 have shown that the abortion bacillus may be isolated from the vaginas of cows for 25 days following abortion or infected full term parturitions. Manthei and Carter3 3 studied the genital infection of 18 experimentally infected cows through 2 pregnancies after exposure, and demonstrated persistent genital elimination of organisms in more than 80% of the animals for at least a month after parturition, and found one cow which shed Brucella for as long as 101 weeks. Brucella abortus was not isolated from the genital tract of 12 of these cows between conception and the second parturition, at which time 3 of them shed the organism. Experiments previously cited indicate that approximately 20% of reactors to the agglutination test are uterine shedders at full term parturitions. Since the first discovery of udder infection in 1911, by Schroeder and Cotton 47 and Smith and Fabyan 4 8 it has been demonstrated by many workers that infected cows may shed Br. abortus from the mammary gland for years. The most recent study of this type is that of Manthei and Carter.33 Fitch et al.49 have demonstrated the organism in urine and feces from infected animals, but concluded that this is not a common occurrence. It would be difficult to rule out contamination by discharges from the reproductive tract. Reference has been made already to elimination of Br. abortus from the male reproductive organs. SEROLOGICAL CHANGES

In animals infected during pregnancy the agglutinin titer generally rises before abortion to 1:200-1:1000 or higher. The incubation period is variable and may last from 1 to more than 30 weeks, depending in part upon the number of organisms.? 0 Some animals may fail to react to the agglutination test until after the termination of the pregnancy, while a few others may fail to react to the agglutination test at all, although definitely infected.30o 31 Even after initial infection a considerable group of animals, which react to the agglutination test in diagnostic titers, are not shedders of infection.? 2 Many of these can be shown to harbor organisms in lymph nodes, and may be considered as "arrested" cases. Some reactors may fail to shed Brucella at a number of parturitions, and then at some later date become persistent shedders. 33 With the passage of time some infected animals cease to react to the agglutination test in diagnostic titers. Beach et al.62 found 14 such ceased reactors in 37 animals within 2 years after experimental infection.

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Winner 5 3 observed an incidence of about 17% in several thousand animals tested in an official eradication program. It has been concludeds 3 that the probability of a ceased reactor being a carrier of Brucella is extremely small. Further Beach et al.64 have shown that recovery from infection, as evidenced by the loss of agglutinins, leaves the animal with a high degree of resistance to reinfection, which it has not been possible to duplicate by vaccination with Strain 19. Other serological changes which have been described during infection are an increase in specific bactericidins66 and a loss of a serum selective factor for smooth organisms. CONCLUSION Examination of the natural course of bovine brucellosis makes untenable the original concept that infection manifests itself as a placental disease, with little or no disturbance of health other than abortion. The research reviewed here shows that infection of cattle with Brucella results in a generalized disease analogous to the disease in guinea pigs, swine and human beings. These facts must be considered in the interpretation of the results of vaccination, and in the establishment of control programs. Real progress in defining the mechanisms of resistance requires that infection per se not be confused with the placental disease. REFERENCES (1) Elberg, S. S. and Silverberg, S. J.: Immunology of brucellosis, Symposium on Brucellosis. Am. Assoc. Adv. Sci., Washington, D. C., 62, 1950. (2) Franck, 1876. Cited by Huddleson.4 2 (3) Lehnert, 1878. Cited by Wilson, G. S. and Miles, A. A.: Topley and Wilson's Principles of Bacteriology and Immunity. Third Edition, Williams & Wilkins Co., Baltimore, 1708, 1946. (4) Bang, B.: Die Aetiologie des Seuchenhaften (Infectiosen) Verwerfens. Zeitschrift fur Tiermedizin 1: 241, 1897. (5) McFadyean, J., Sheather, A. L., and Minett, F. C.: Researches regarding epizootic abortion of cattle. Jour. Comp. Path. 26: 142, 1913. (6) Seddon, H. R.: Studies in abortion disease. Jour. Comp. Path. 32: 1, 1919. (7) King, R. O. C.: Brucella infection in the bull: A progress report of mating experiments with naturally infected bulls. Australian Vet. Jour. 16: 117, 1940. (8) Bendixen, H. C.: Et Filfaelde af akut Brucellainfecktion i Pars glandularis ductus deferentis hos Tyr, Maanedsskr. Dyrl. 66: 1, 1944. (9) Seit, B.: Kastningssmitte ved kunstig Inseminering, Maandsskr. Dyrl. 56: 12, 1944. (10) Manthei, C. A., DeTray, D. E., and Goode, E. R.: Brucella infection in bulls and the spread of brucellosis in cattle by artificial insemination. I. Intrauterine injection, cited in Manthei.21 (11) Christensen, N. O.: Studies on the agglutinin formation in brucellar infection of the genitals of the bull. Acta Path. et Microbiol. Scand. 25: 202, 1948. (12) Bang, B.: Infectious abortion in cattle. Jour. Comp. Path. 19: 191, 1906. (13) Cameron, H. S.: The vitality of Br. abortus. Cornell Vet. 22: 212, 1932.

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(14) Bosworth, T. J.: Fourth Rep. Director Inst. Anim. Path., Cambridge 65: 1934-35. (15) Bang, O. and Bendixen, H. C.: Does the Br. abortus go through the normal hide and is this an important way of infection in cattle? Saertryck af Medlemsblad for Den danske Dyrlaeg. 15: 1, 1932. (16) Thomsen, A.: Antibody production in infectious abortion, especially in cattle. Skand. Veterinaertidsk. 8: 233, 1918. (17) Tovar, R. M.: Infection and transmission of Brucella by ectoparasites. Amer. Jour. Vet. Res. 8: 138, 1947. (18) Washko, F. V., Hutchings, L. M. and Donham, C. R.: Studies on the pathogenicity of Brucella suis for cattle. Amer. Jour. Vet. Res. 9: 342, 1948. (19) Elberg, S. S. and Henderson, D. W.: Respiratory pathogenicity of Brucella. Jour. Inf. Dis. 82: 302, 1948. (20) Berman, D. T., Irwin, M. R. and Beach, B. A.: Statistical considerations of controlled experiments in brucellosis. Amer. Jour. Vet. Res. 10: 130, 1949. (21) Manthei, C. A.: Brucellosis in cattle, Symposium on Brucellosis. Am. Assoc. Adv. Sci., Washington, D. C., 172, 1950. (22) Edgington, B. H. and Donham, C. R.: Infection and reinfection experiments with Bang's disease. Jour. Agric. Res. 59: 609, 1939. (23) Bang, O., Bendixen, H. C. and Perskov, J.: Ruft Brucella abortus Bang eine universelle Infektion beim Rinde hervor? Acta Path. et Microbiol. Scand., Suppl. 16, 7, 1933. (24) Berman, D. T.: Unpublished data, 1950. (25) Carpenter, C. M.: Bacterium abortus invasion of the tissues of calves from the ingestion of infected milk. Cornell Vet. 14: 16, 1924. (26) Mitchell, C. A. and Gwatkin, R.: The relationship of oestrin to colonization of Brucella abortus in calves. Canad. Jour. Comp. Med. 9: 16, 1945. (27) Schroeder, E. C. and Cotton, W. E.: Some facts about abortion disease. Jour. Am. Vet. Med. Assoc. 50: 321, 1916. (28) Birch, R. R. and Gilman, H. L.: The agglutination test in relation to the persistence of Bacterium abortus in the body of the cow. Ann. Rept. N. Y. State Vet. Coll., Cornell Univ., Ithaca, N. Y., 56, 1931. (29) Roots, E. and Ridala, W.: Untersuchungen iber das Wesen der Brucellose u. uber die Ansiedlungsorte der Br. abortus im Korper des Rindes, in besondere in der Thyreoidea, Eesti Loomaarst 8: 165, 1932. (30) Krtger, H.: Ueber das Vorkommen von Bang bacterien im Fleisch geschlacteter Rinder, Deuts. Tierárztl. Wschr. 40: 481, 1932. (31) Doyle, T. M.: The distribution of Br. abortus in the system of "carrier" cows. Jour. Comp. Path. &Therap. 48: 192, 1935. (32) Berman, D. T.: Unpublished data, 1946. (33) Manthei, C. A. and Carter, R. W.: Persistence of Brucella abortus infection in cattle. Am. Jour. Vet. Res. 11: 173, 1950. (34) Soule, M. K.: Bacteriological and serological findings in Brucella abortus infections in animals and man. Premier Congrés International de Mi crobiol. 1: 606, 1930. (35) Fitch, C. P., Bishop, L. M. and Kelly, M. D.: The isolation of Brucella abortus from the blood stream of cattle. Proc. Soc. Exp. Biol. & Med. 34: 696, 1936. (36) Fitch, C. P., Boyd, W. L., Bishop, L. M. and Kelly, M. D.: Localization of Brucella abortus in the bovine uterus. Cornell Vet. 22: 62, 1939. (37) Gwatkin, R.: Incidence of Brucella abortus in the fetal membranes of fulltime, reacting cows. Cornell Vet. 22: 62, 1932.

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38) Fitch, C. P., Boyd, W. L. and Bishop, L. M.: A study of the vaginal content of pregnant Bang infected cows for the presence of Brucella abortus. Jour. Amer. Vet. Med. Assoc. 92: 171, 1938. (39) Hallman, E. T.: Further studies in the reproductive organs of cattle. Cornell Vet. 14: 254, 1924. (40) Ridala, V.: Inquiries into the pathogenic changes produced by Brucella abortus in the udder and certain organs of the cow. Vet. & Milk Hyg. Inst., Univ. Tartu, Estonia, 1936. (41) Runnells, R. A. and Huddleson, I. F.: The nature of Bacterium abortus infection in the udder of the bovine. Cornell Vet. 16: 376, 1925. (42) Huddleson, I. F.: Brucellosis in man and animals. The Commonwealth Fund, New York, 1943. (43) Berman, D. T. and McNutt, S. H.: The reaction of the reticulo-endothelial system in naturally acquired bovine brucellosis. Unpublished data, 1947. (44) Brown, I. W., Forbus, W. D. and Kerby, G. P.: The reaction of the reticulo-endothelial system in experimental and naturally acquired brucellosis of swine. Amer. J. Path. 21: 205, 1945. (45) Sundberg, R. D. and Spink, W. W.: The histopathology of lesions in the bone marrow of patients having active brucellosis. Blood, Suppl. 1, 1947. (46) Fitch, C. P., Delez, A. L. and Boyd, W. L.: Duration of the elimination of Bacterium abortus in the vaginal and uterine discharges of infected cattle. Jour. Am. Vet. Med. Assoc. 76: 680, 1930. (47) Schroeder, E. C. and Cotton, W. E.: The bacillus of infectious abortion found in milk. 28th Ann. Rept. Bur. Ani. Ind., U. S. D. A., 139, 1911. (48) Smith, T. and Fabyan, M.: The pathogenicity of Bacillus abortus Bang. Centralblatt fur Bact. (Abt 1) 61: 549, 1911. (49) Fitch, C. P., Bishop, L. M. and Boyd, W. L.: A study of bovine blood urine and feces for the presence of Bacterium abortus Bang. Proc. Soc. Exp. Biol. & Med. 29: 555, 1932. (50) McEwen, A. D., Priestly, F. W. and Patterson, J. D.: An estimate of a suitable infective dose of Br. abortus for immunization test on cattle. Jour. Comp. Path. & Therap. 62: 116, 1939. (51) Birch, R. R., Gilman, H. L. and Stone, W. S.: The immunity created by vaccination of calves with Brucella abortus Strain 19. Cornell Vet. 35: 119, 1945. (52) Beach, B. A., Irwin, M. R. and Ferguson, L. C.: The significance of the "ceased" reactor to Bang's disease. Jour. Agric. Res. 61: 75, 1940. (53) Winner, W.: Personal communication, 1948. (54) Beach, B. A., Irwin, M. R. and Ferguson, L. C.: The response of "ceased" reactors in Bang's disease to reexposure. Jour. Agric. Res. 66: 523, 1942. (55) Irwin, M. R. and Berman, D. T.: Bactericidal tests in brucellosis, Symposium on Brucellosis. Am. Assoc. Adv. Sci., Washington, D. C., 85, 1950. (56) Braun, W.: Variation in the genus Brucella, Symposium on Brucellosis. Am. Assoc. Adv. Sci., Washington, D. C., 26, 1950. LA EVOLUCIóN NATURAL DE LA BRUCELOSIS BOVINA (Sumario) Se discute la infección del ganado con Brucella abortus tomando en cuenta las vías de invasión, y el análisis estadístico de los coeficientes de infección en el ganado infectado experimentalmente. Se analiza también la influencia de distintas cantidades de microorganismos sobre el coeficiente de infección en las

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pruebas experimentales. Un repaso de los estudios de la distribución del microorganismo en el huésped recalca la importancia de las infecciones extragenitales. Los tejidos extragenitales invadidos con más frecuencia son los ganglios linfáticos, el bazo y el hígado, asi como la glándula mamaria. La enfermedad se caracteriza también por bacteriemia intermitente, en particular durante las fases agudas. El examen histopatológico de los nódulos linfáticos infectados revela alteraciones granulomatosas que se caracterizan por proliferación de los reticulocitos e infiltración con plasmocitos y monocitos, y en ocasiones formación de células gigantes, pero con poca necrosis. Se conoce más generalmente la necrosis y exudado descubiertos en la placenta y en las carúnculas uterinas. El microorganismo se elimina principalmente en las excreciones del aparato de la reproducción durante el parto, y durante períodos variables después. En la transmisión de la infección al hombre, la introducción del microorganismo en la leche reviste mayor importancia que su diseminación entre el ganado. Los toros con infección del aparato de la reproducción pueden eliminar brucelas en el semen. La infección bovina se caracteriza por la elaboración de aglutininas específicas, anticuerpos que fijan el complemento y aumento de la actividad bactericida específica del suero. También se ha descrito la pérdida de un factor del suero que fomenta el establecimiento de una brucelal lisa. Se discuten la frecuencia y el significado inmunológico de la reposición espontánea.

THE NATURAL COURSE OF SWINE BRUCELLOSIS* By L. M. HUTCHINGS, B.S., D.V.M., M.S., Pn.D. Although swine brucellosis has been discussed many times in the past thirty-five years, it seems appropriate to again review the more important aspects of the natural course of the disease at the Third InterAmerican Congress on Brucellosis. Since 1941, when Dr. Jacob Traum first reported the recovery of Brucella organisms from aborted swine fetuses there have been numerous investigations dealing with many phases of swine brucellosis. Unfortunately, however, we are unable to draw upon the vast volume of investigational work which has been reported on bovine brucellosis. It is now recognized that swine brucellosis, although not of paramount economic significance to the pork producer, is often a serious economic problem to the professional swine breeder. In addition to the economic loss to the swine industry, brucellosis of swine is a menace to human health and to the health of other species of animals. Knowledge has recently accumulated concerning naturally occurring cases of Brucella abortus and Brucella melitensis infection in hogs (McCullough et al., 1949; Jordan and Borts, 1946). Thus further emphasis may be placed on swine as a reservoir for human brucellosis, since any species of animal which has been shown to harbor all three species of Brucella must be regarded as a potential source of spread. Our understanding of the precise means by which Brucella infection spreads from swine to cattle and man is not complete, but reports of the recovery of Br. suis from naturally infected cow's milk have been made by Hasseltine (1930), Beattie and Rice (1934) and Borts et al. (1943). Washko et al. have demonstrated experimental infection of cattle by intramammary exposure to Br. suis. Several reports have shown that Brucella may be found in freshly dressed pork and that these organisms may survive for relatively long periods of time (up to at least 20 days) under refrigeration. Thus man could acquire brucellosis by contact with this infected meat as well as by contact with the live infected hog. From this brief introduction, it can be readily understood that an infection cycle between swine, cattle and man is of considerable importance, not only to the industries involved, but also to the epidemiology of brucellosis in human beings. Since brucellosis generally is not transmitted from man to man, domestic animals are regarded as the chief source of human infection. Therefore the control of the disease in man is largely dependent upon the control of brucellosis in animals. * Published as Journal Series Paper No. 475 of the Purdue University Agricultural Experiment Station, Lafayette, Indiana.

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This makes the problem of brucellosis control of vital importance to the veterinary profession. In the available time, an attempt will be made to discuss briefly the natural course of swine brucellosis from the standpoints of susceptibility, transmission, symptoms, resistance and therapy, diagnosis, prevention and control. SUSCEPTIBILITY

Swine of both sexes and all ages appear to be susceptible to brucellosis. Symptoms are more pronounced in adult swine than in pigs. Suckling pigs seem to be less susceptible than weanling pigs, but the suckling pig may be infected either artificially or under field conditions. These observations are similar to those made on cattle and human beings as far as age susceptibility is concerned. It is a common belief that pregnancy or sexual maturity are essential for infection of swine with Brucella. Observation and experience have shown this belief to be erroneous. There is ample evidence that males are as susceptible as females. Castrates may acquire the disease also. In considering susceptibility, it should be pointed out that swine of all ages and both sexes appear to have some natural resistance to brucellosis. This is indicated by the size of exposure doses usually employed in infection of swine experimentally and by the difficulties commonly encountered in experimental production of abortions. TRANSMISSION

There are some features of the spread of brucellosis which are not well understood in swine as in other species. However, from the available information, the author considers swine brucellosis to be spread primarily by animal to animal contact and secondarily by indirect means, such as contaminated transportation facilities, carrier animals and birds, contaminated feed sacks and the innumerable other methods of indirect contact. Transmission may occur by ingestion of infected material, contact with infected discharges, by breeding, transportation, shows, sales, fairs and probably many other ways. An infected boar may eliminate Brucella in the semen in tremendous numbers. Thus the infected boar is often observed to be the cause of spread, especially when introduced into a herd which is free of brucellosis. Although adult or breeding swine are most commonly considered in the spread of brucellosis, one must not overlook the possibility of spread from infected pigs. It is known that Brucella may localize in many if not all parts of the body and hence elimination of the organisms may occur in any of the natural body discharges such as milk, urine, and

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feces in addition to semen. Infected sows which abort or farrow normally may eliminate Brucella in the urogenital discharges for variable intervals after parturition. Br. suis is prone to produce abscesses; these abscesses may rupture from time to time and spread Brucella about the premises. The livability of Brucella suis outside the animal body has not been extensively studied, but must be considered in transmission of brucellosis. Such studies as have been made indicate that Br. suis may live for three or four months in such materials as tap water and sterile soil, but for shorter periods of a month or so in distilled water, urine, feces, and unsterile soil. SYMPTOMS

The variation in symptomatology which is observed in swine brucellosis is somewhat dependent upon the localization of the organisms in the body. Following exposure to Br. suis, swine often develop a bacteremia which persists for variable periods of time, usually not more than 60-90 days. During and subsequent to the bacteremia, the organisms tend to localize. The points of localization are unpredictable and Br. suis has been found in most if not all of the tissues and organs of infected swine. The lymphatic tissue and sexual organs are common sites for localization; but isolation has been made from liver, brain, spinal cord, joints, kidney, bladder, mammary tissue and other portions of the body. The gross symptoms most commonly observed are abortion, sterility, orchitis, lameness, posterior paralysis, and occasionally metritis and abscessation of the extremities or other parts of the body. The symptom of abortion is rather variable, hence it is extremely difficult to predict the number of abortions in any Brucella infected herd. The abortion rate may be 50 to 80% of the females, or, in other herds which are equally infected, as measured by the serum agglutination test, there may be no observed abortions. The stage of gestation at which infection occurs, the virulence of the organism, and the susceptibility of the swine probably all play a part in the outcome of pregnancy. Abortions may occur at any time during pregnancy; those which occur very early in gestation are often unobserved. The symptoms of lameness, posterior paralysis and abscessation are commonly associated with the localization of Brucella in the areas involved. Sterility in gilts, sows and boars is common and sometimes is the only outward sign of brucellosis in the herd. Sterility in the female is often temporary. Orchitis in the boar is frequently observed where brucellosis exists. The orchitis is usually unilateral. Sterility may or may not occur as a result of orchitis. Fertility appears to be lowered, but complete sterility may not be present.

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Brucellosis of swine appears to be a somewhat self limiting type of disease at least in so far as abortion and serum agglutination are concerned. These features of the disease have contributed to the reports claiming the development of a serviceable immunity through the use of live or killed vaccines.This has been especially true when the results of vaccination have been measured by breeding efficiency only rather than by the more rigid criterion of freedom from infection. There is ample evidence that a tolerance to Brucella is developed by swine through natural acquisition of brucellosis or through vaccination. It has been demonstrated many times that most female swine abort only once, a few may abort twice and very few become habitual aborters as a result of Brucella infection. This in itself is an indication of tolerance to the infectious agent. As yet investigators have not satisfactorily exploited such natural phenomena by means of vaccination to a point where the artificial production of immunity is considered practical. Chemotherapeutic and antibiotic agents and combinations of the two have been studied to a limited extent, but the results to date have not been sufficiently encouraging to justify field usage. From the brief summary of resistance and therapy for swine brucellosis, it seems obvious that control of the disease rests largely in our ability to diagnose the infection and then make suitable disposition of the infected herds and animals. DIAGNOSIS

The principal method of diagnosis of swine brucellosis in current use is the serum agglutination test, the same as for the diagnosis of bovine brucellosis. The complement fixation test and various intradermal allergic tests have been tried but have been discontinued as too cumbersome or less accurate than the standard agglutination test. It is generally accepted that the serum agglutination test in swine is effective in determining the presence or absence of brucellosis in the herd, but has its limitations in detecting brucellosis in individual animals. Therefore it is necessary to use the agglutination test for diagnosing the disease in a herd and to base attempts at control on entire herds or units rather than on individual swine. It is now considered necessary to conduct the test in serum dilutions of 1:25, 1:50, 1:100 and above. The interpretation of the test requires some judgement since in nearly any sizeable herd of swine low-titered reactions occur in the absence of demonstrable infection. As a practical rule serum agglutination reactions below the 1:100 dilution are not considered indicative of brucellosis unless there are definite reactors at the 1:100 dilution or higher in the herd. Purchase of individual swine which exhibit a low-titered agglutination response should be avoided unless the status of the entire herd of origin is known.

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It is beyond the scope or time allotted to this paper to discuss the details of control other than to point out that two methods have been successful. 1. Test and prompt sale for slaughter of the entire herd if infection is detected. 2. Test, segregation, and delayed slaughter of the infected herd. The essential steps in this plan are: (a) Blood test the entire breeding herd. (b) If infection is present, consider the entire herd as infected, rather than remove the positively reacting animals. Manage the herd as a unit. (c) Raise the pigs from this infected unit. Wean and test the pigs at 8 weeks of age. Isolate the negative pigs on clean premises as far removed as possible from the infected parent herd. Maintain this isolation until it is practical to dispose of the infected parent herd. (d) Blood test the pigs up to, and during, the first pregnancy. Remove all reactors as they occur. Breed only those gilts which are negative to the blood test to non-infected boars. (e) Dispose of the original infected herd as soon as suitable negative replacements are available, or as soon as it is obvious that the plan is giving satisfactory results. (f) Premises where the infected herd was kept should be cleaned and disinfected throughly prior to admission of the clean replacement herd. Citations to the literature have been kept to a minimum for purposes of clarity and conservation of space; however, a partial bibliography appears at the end of this discussion. BIBLIOGRAPHY Beattie, C. P., and Rice, R. M. (1934): Undulant fever due to Brucella of the porcine type Brucella suis. Jour. Am. Med. Assoc., 102: 1670. Borts, I. H., Harris, D. M., Joynt, M. F., Jennings, J. R., and Jordan, C. F. (1943): A milk-borne epidemic of brucellosis. Jour. Am. Med. Assoc., 121: 319. Borts, I H. (1945): Some observations regarding the epidemiology, spread and diagnosis of brucellosis. Jour. Kansas Med. Soc. Dec., 1945, pp. 1-7. Cameron, H. S. (1943): Brucellosis in swine. I. The interpretation of low titer reactions in experimental and field infections. Am. Jour. Vet. Res., 4: 169-172. Cameron, H. S., Gregory, P. W., and Hughes, E. H. (1943): Inherited resistance to brucellosis in inbred Berkshire swine. Am. Jour. Vet. Res. 4: 387-389. Cameron, H. S. (1947): Brucellosis eradication and its effect on production in a large swine herd. Cornell Vet., 37: 55-58. Connaway, J. W., Durant, A. J., and Newman, H. G. (1921): Infectious abortion in swine. Missouri Agr. Expt. Sta. Bull., 187. Cotton, W. E., Buck, J. M., and Smith, H. E. (1938): Communicability of infectious abortion between swine and cattle. U. S. D. A. Tech. Bull., 629. Graham, Robert, Boughton, I. B. and Tunnicliff, E. A. (1930): Studies on porcine infectious abortion. Univ. Illinois Agr. Expt. Sta. Bull., 343. Hadley, F. B., and Beach, B. A. (1922): An experimental study of infectious abortion in swine. Univ. Wisconsin Agr. Expt. Sta. Res. Bull., 55.

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Harms, Amanda H. (1932): Cattle as a possible source of infection from Brucella abortus var. suis. Jour. Am. Vet. Med. Assoc., 34: 246-249. Hashizumo, K., Kamoto, K., and Kurizawa, T. (1940): Swine brucellosis. Res. Bull. Imp. Zooteck., Expt. Sta. Tibasi, 42: 1-113. Hasseltine, H. E. (1930): The relationship of human and animal brucellosis. Jour. Am. Vet. Med. Assoc., 29: 330-338. Hutchings, L. M. (1943): Brucellosis in swine. Proc. 47th Annual Meeting U. S. Livestock Sanitary Assoc., pp. 52-58. Hutchings, L. M., Delez, A. L., and Donham, C. R. (1944): Studies on brucellosis of swine. I. Infection experiments with weanling pigs. Am. Jour. Vet. Res., 5: 195-208. Hutchings, L. M. (1944): Report of further studies of brucellosis in swine. Proc. 48th Annual Meeting U. S. Livestock Sanitary Assoc., pp. 105-109. Hutchings, L. M., and Andrews, F. N. (1946): Studies on brucellosis in swine. III. Brucella infection in the boar. Am. Jour. Vet. Res., 7: 379-384. Hutchings, L. M. (1947): Field control experiments with brucellosis in swine. Proc. 51st Annual Meeting of U. S. Livestock Sanitary Assoc., pp. 124-136. Johnson, H. W., and Huddleson, I. F. (1931): Natural Brucella infection in swine. Jour. Am. Vet. Med. Assoc., 78: 849-862. Jordan, Carl F. (1942): Undulant fever in Iowa. Proc. 46th Annual Meeting of the U. S. Livestock Sanitary Assoc., Chicago. Jordan, C. F., and Borts, I. H. (1946): Occurrence of Brucella melitensis in Iowa. Jour. Am. Med. Assoc., 130: 966. Kernkamp, H. C. H. (1942): Forum on infectious and transmissible diseases of swine. Proc. 46th Annual Meeting U. S. Livestock Sanitary Assoc., p. 63. Kernkamp, H. C. H., and Roepke, M. H. (1948): The interpretation of low agglutination titers in the control of swine brucellosis. Am. Jour. Vet. Res., 9: 46-49. Manthei, C. A. (1948): Research on swine brucellosis by the Bureau of Animal Industry (1941-1947). Am. Jour. Vet. Res., 9: 40-45. McCullough, N. B., Eisele, C. W., and Pavelchek, Emma (1949): Isolation of Brucella abortus from hogs. Pub. Hlth Repts., 64: 537-538. McNutt, S. H. (1935): Incidence and importance of Brucella infection of swine in packing houses. Jour. Am. Vet. Med. Assoc., 86: 183-191. McNutt, S. H. (1938): Brucella infection in swine. Proc. 42nd Annual Meeting of the U. S. State Livestock Sanitary Assoc., Chicago. Moore, V. A. (1929): Relation of undulant fever in man to livestock sanitation. J. Am. Vet. Med. Assoc., 27: 605-617. Stone, W. S. (1943): Brucellosis in swine. Cornell Vet., 83: 115-119. Thomsen, Axel. (1934): Brucella infection in swine. Acta Path. Microbiol. Scand. Suppl. 21. Translation by Hans Anderson, M.D. Washko, F. V., Hutchings, L. M., and Donham, C. R. (1948): Studies on the pathogenicity of Brucella suis for cattle. I. Am. Jour. Vet. Res., 9: 342-350. EL CURSO NATURAL DE LA BRUCELOSIS PORCINA (Sumario) En la actualidad, la brucelosis porcina está reconocida como una enfermedad impotante, tanto bajo aspectos económicos como de salud pública. Se ha demostrado que el cerdo es susceptible a las tres espeies de Brucella, esto es, Br. abortus, suis, y melitensis.

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El hombre puede contraer la brucelosis por contacto, ya con cerdos vivos infectados o, potencialmente, por contacto con carnes de cerdos infectados. Se ha demostrado que la Brucella permanece activa por lo menos 20 dfas en los tejidos del cerdo infectado en forma natural. Los síntomas de brucelosis observados con más frecuencia en los cerdos son: aborto, esterilidad, lisiamiento, orquitis, parálisis posterior y abscesos. Los sfntomas dependen en parte de la localización de los organismos. Los cerdos de toda edad y de ambos sexos son susceptibles, pero los efectos son más graves en animales adultos. La transmisión ocurre principalmente por contacto directo de animal con animal. La propagación puede ocurrir indirectamente a través de medios de transporte, alimentos contaminados, pocilgas y, quizás, hasta por agentes mecánicos. La Brucella puede ser eliminada en la leche, orina, heces, semen y exudaciones urogenitales de los cerdos infectados. El diagnóstico está basado en la interpretación de los resultados de la prueba normal de aglutinación del suero, tal como sucede en la brucelosis bovina. Esta prueba es exacta cuando se toma procedimiento de diagnóstico en un rebaño, pero tiene ciertas limitaciones para el diagnóstico individual de la enfermedad en el cerdo. El control de la brucelosis porcina depende del diagnóstico, así como de ciertas formas de segregación o eliminación de cerdos infectados o de rebaños enteros. La vacunación y el tratamiento no han demostrado aún ser satisfactorios para el control de la brucelosis porcina.

STUDIES OF THE PHYSICAL PROPERTIES AND AGGLUTINABILITY OF BRUCELLA ANTIGENS USED IN THE AMERICAS* By FRED M. MURDOCKf, MARTIN H. ROEPKES AND BENJAMIN D. BLOOD§ The blood serum agglutination reaction is a relatively simple test widely used for the diagnosis of brucellosis in both animals and man. Unfortunately the antigens and techniques for this test have not been standardized on an international basis. The results have thus been subject to controversy and misinterpretation. This situation was recognized in the recommendation of the XII Pan American Sanitary Conference (Caracas-1947), "that the methods and means of brucellosis diagnosis be made uniform throughout the American Republics". The Second Inter-American Congress on Brucellosis (Buenos Aires-1948) also emphasized the need for standardization of diagnostic methods for human and animal brucellosis and recommended that the agglutination reaction with standard antigen be employed. The purpose of the present studies was to determine the similarities or variances that existed between Brucella antigens used in the Americas. The Pan American Sanitary Bureau obtained the antigens for this study by arrangements with various governmental laboratories and agencies concerned with serum agglutination tests for the diagnosis of brucellosis. The antigens were received from countries throughout the Americas; in addition, several antigens produced in Europe were included. Thirty-six antigens, including seventeen for the tube test and nineteen for the plate test, from twenty-five governmental and private laboratories in fifteen different countries and territories, were studied and are included in this report. Governments which cooperated in the project were those of Argentina, Brazil, Canada, Chile, Cuba, Denmark, Dominican Republic, Ecuador, Great Britain, Jamaica, Mexico, The Netherlands, Peru, United States and Venezuela. Brucella Species.-The species of Brucella used in the preparation of those antigens for which information was supplied, were as follows: * Paper No. 2608, Scientific Journal Series, Minnesota Agricultural Experiment Station, University of Minnesota. This study was supported by a grant from the Pan American Sanitary Bureau. t United States Bureau of Animal Industry, St. Paul, Minnesota. At present

employed by the Anchor Serum Co., South St. Joseph, Missouri. : University of Minnesota, St. Paul, Minnesota. § Pan American Sanitary Bureau, Washington, D. C. 122

PHYSICAL PROPERTIES OF BRUCELLA ANTIGENS Brucella Brucella Brucella Brucella Brucella

abortus ......................................... suis ............................................. melitensis ....................................... abortus and Brucella suis ........................ abortus and Brucella melitensis ..................

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19 antigens 2 antigens 2 antigens 1 antigen 2 antigens

Culture Media.-For the preparation of the several antigens, the Brucella cultures were grown as follows: Medium

No. Antigens

Potato agar ..................................................... 14 Liver infusion agar .............................................. 4 Peptone agar .................................................... 6 Tryptone agar .................................................. 3 Glucose agar .................................................... 2 Dextrose agar ................................................... 2 Hartly digest agar .............................................. 1 Medium prepared from beef broth and peptone with dextrose & glycerine ..................................................... 1 Medium prepared from meat infusion and peptone ............... 1

Growth Period.-The period of growth of the cultures was 48 hours for 15 antigens, 72 hours for 18 antigens and 96 hours for 1 antigen. Harvesting Solutions.-The growth was harvested with: 0.5% phenol in physiological salt solution ................. 13 antigens 0.5% formalized physiological salt solution ............... 6 antigens Physiological salt solution ................................ 5 antigens 12% salt solution ......................................... 4 antigens Physiological salt solution containing 1% formalin ........ 2 antigens 2 antigens 1.2% salt solution, containing 0.5% phenol ............... 12% salt solution plus 1% phenol ........................ 1 antigen Physiological salt solution plus 1% glycerine and 0.25% phenol .............................................. 1 antigen

Killing and preservation.-In the majority of instances the bacteria were killed by heating the suspensions to temperatures ranging from 60 to 100°C for periods ranging from 10 minutes to 60 minutes, or by boiling for 10 to 30 minutes. In 6 instances, the organisms were killed with 0.5% formalin, 2 by undiluted formalin and 1 by 95% alcohol. For a high percentage of the antigens, the killed cells were suspended in a sodium chloride solution of concentrations ranging from 0.85% to 1.2% and containing as a preservative 0.5% phenol. In some instances, small amounts of dyes, such as crystal violet and/or brilliant green, were added. In 2 instances, 1% formalin plus merthiolate were added as preservatives; in 1 instance, 1% phenol, and in another, 0.25% formaldehyde, for the same purpose. Cell concentration.-Standardization of the density or concentration of cells in 8 cases depended upon the McFarland nephelometer, in 7

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cases, upon the per cent by volume of centrifuged cells as determined by the Hopkins vaccine tube or the Fitch-modified-Hopkins vaccine tube; in 4 instances upon testing with serum of known titer; in 2 upon the Fitch-modified-Hopkins vaccine tube and the testing with sera of known titers; in 4 upon comparison with former lots of antigen prepared in the laboratory; in 1, upon comparison with antigen from another country and testing with sera of known titers; in 1 upon an absorptiometer; and in 1, upon comparisons with another antigen. Intended use.-The species for which the antigens were to be used were designated as follows: Human ............................................................ Bovine ............................................................. Bovine, porcine and human ........................................ Porcine. ........... ......................................... Bovine and porcine ................................................ Porcine, caprine and human ........................................ Bovine, porcine and caprine ........................................ Melitensis infections ...............................................

5 4 2 2 2 1 1 2

TUBE TEST METHODS A summary of the instructions given for conducting the tube agglutination test follows: Dilutions.-The volume of the serum-antigen mixture was 1 cc for 11 of the antigens and 2 cc for the remaining 7. The diluent to be used for the tube test was 0.5% phenol in physiological salt solution for 9 of the antigens, and plain sterile physiological salt solution for 8 of the antigens. The tests were to be incubated. Incubation Period.At 37°C for 72 hours, 6 antigens; for 48 hours, 2 antigens; for 24 hours plus 8 hours at room temperature, 1 antigen; for 24 hours plus 24 hours at room temperature, 1 antigen; for 24 hours plus over night at room temperature, 1 antigen; for 18 hours plus 20 to 24 hours at room temperature, 1 antigen; for 20 hours, 2 antigens; for 2 hours plus 22 hours in the refrigerator plus 2 to 3 hours at room temperature, 1 antigen; for 2 hours plus over night in the refrigerator, 1 antigen; At 56°C for 20 to 25 hours in a water bath, 1 antigen. Reading Instructions.-In almost all instances, the instructions with respect to the lighting arrangement involved a shaded light for good illumination of the specimens and a dark background, for visual reading of the degree of agglutination. In one instance a low power magnifying lens was recommended for better observation of the agglutination re-

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action. The instructions for the reading of the tests, in a high percentage of the cases, recommended designating the degree of agglutination for each dilution as negative, partial or complete agglutinations. In a few instances, it was recommended that the degree of reaction be graded from negative to 3 or 4+, for each dilution in the test. PLATE TESTS

With respect to the plate test, recommendations for the time of reading were: 15 minutes for 4 antigens 8 minutes for 3 antigens 5 minutes for 3 antigens 3 minutes for 2 antigens 8 to 10 minutes for 1 antigen 5 to 8 minutes for 1 antigen 2 minutes for 1 antigen and one antigen, after rocking the plate back and forth 15 to 20 times. LABORATORY COMPARISON OF ANTIGENS

Laboratory studies made on the antigen included the following procedures: microscopic and cultural examinations for purity; concentration of the antigens, in terms of the volume of centrifuged cells per 100 cc, as determined by centrifugation at 2800 r.p.m. for 75 minutes with the Fitch-modified-Hopkins vaccine tube, and agglutination tests on 55 bovine and caprine serums of various titers to determine agglutinability and sensitivity of the antigens. Purity.-The per cent of extraneous cells in the antigen, as determined by microscopic examination of stained specimens, varied from 0 to 13%. The bacterial count of viable cells per cc of plate antigen, or concentrated tube antigen as received, was in 18 cases 0; in 12 antigens, the counts ranged from 1000 to 8500; in 5 antigens, from 51,000 to 84,000; in 1 antigen, 360,000, and in 3 antigens, 3 to 10 million. The viable cells found were not Brucella. As near as could be determined from observations on agglutinability with the 55 sera used, the extraneous cells found did not materially interfere with the agglutination reaction. Agglutinability.-The bovine sera with low to high titers used for the agglutination studies were obtained either from known infected herds, or from animals located in the stockyards at packing plants. The sera with negative titers were obtained from a negative herd. The titers of the serum ranged from negative at the 1:25 dilution to complete agglutination at 1:1600 as determined by the tube method of the United States Bureau of Animal Industry. The titers of the 24 sera used to

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calculate the relative sensitivity of the antigens, ranged from incomplete or partial agglutination at the 1:50 dilution, up to partial agglutination at 1:200. The instructions supplied with each of the antigens were followed as closely as possible with respect to the preparation of the test, period of incubation and the reading of the test. Tests were read in such a way that the operator had no knowledge of the number of the serum concerned, in order to insure unbiased readings. Table I summarizes observations on the antigens studied. Under the columns with headings, "Country", and "Laboratory", respectively, Roman numerals designate countries; capital letters designate the laboratories or private concerns which submitted the respective antigens; the Arabic numerals indicate the different antigens from each laboratory. Antigen concentration is in terms of per cent by volume of centrifuged cells. Concentrations are listed for the final dilution of the antigen as present in the test. In the case of plate test antigens, concentrations are those found in the antigens as received ready for use. The relative sensitivity of the antigen, as given in the sixth column, is numerically the average titer obtained on 24 sera in the low and intermediate titer range. The average titer obtained for an antigen with the 24 sera was calculated as follows: each dilution of a single test was given a value of 1 for complete agglutination, 0.5 for partial or incomplete agglutination, and 0 for no agglutination. Thus, with a test starting at the 1:25 dilution, a titer on a serum showing incomplete agglutination at 1:100 was given a titer value of 2.5. A serum showing complete agglutination at 1:50 was given a titer value of 2. Likewise, a serum showing incomplete or partial agglutination at 1: 50 was given a value of 1.5, and so on. By adding the titer values of each of the 24 sera and dividing by 24 the average titer value was obtained. This average titer value was then reconverted to an average titer. For example: an average titer value of 2.68 is equivalent to complete agglutination at a dilution of 1 to quantity 50, plus 68% of the difference between 50 and 100, or complete agglutination at 1:84. The relative sensitivity would then be 84. The method of determining the relative sensitivity of antigens for comparative purposes, by conducting tests on 20 to 30 sera of low to intermediate titers, with unbiased readings of the test, has been found to be very satisfactory. The relative sensitivity values obtained as noted may be duplicated within plus or minus 10%, in a very high percentage of cases. If small differences in antigen sensitivity are to be accurately evaluated, an appreciable equal number of sera must be used, and readings must be entirely independent. The next column lists the minimal significant titer where instructions included the interpretation of the agglutination reactions. In those instances in which the minimal significant titer was given as partial agglutination at a given dilution, the significant titer was designated as

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midway to the next lower dilution. For example, a significant titer or partial agglutination at 1:100 is designated as equivalent to complete agglutination at 1:75, i.e., midway to the next lower dilution. Likewise, partial agglutination at 1:80 is considered equivalent to complete agglutination at 1:60, and partial agglutination at 1:40 equivalent to complete agglutination at 1:30. This was'done to facilitate comparisons of the various antigens and calculation of "relative applied sensitivities". The eighth column contains calculated values of "the relative applied sensitivity". These values combine the factors of (a) relative sensitivity and (b) significant titer. They were computed by dividing the relative sensitivity value by the significant titer value and multiplying the quotient by 100. Since these values combine the relative sensitivity and the interpretations of the agglutination test, they indicate the sensitivity of the test as applied under field conditions. In Table I, under "Interpretation" are interpretations of the 24 sera tested, in terms of significant, doubtful and negative titers. Under "Readability of Test," subjective evaluations are given with respect to the ease of reading the test. These evaluations were made from the standpoints of character of agglutination and sharpness of end-point. The agglutination reactions obtained with antigens XI A-1 and XII A-2 were so erratic that relative sensitivity calculations could not be made. With these two antigens, the tests were negative on a number of the high titered sera. Antigen XII A-1 is a special type of antigen to be used with a special technic of conducting the test, using one dilution. The results of agglutination reactions obtained with this special test appeared to be quite satisfactory from the standpoint of separating sera giving titers above 1:100 with the majority of the antigens from those below 1:100. In several instances, information was not given with respect to significant titers. The relative applied sensitivity and interpretation values could thus not be determined in these cases. In two instances, insufficient amounts of antigen were supplied to conduct the test on all of the 24 sera used for sensitivity determinations. For these, values could not be given in the "Interpretation" column. Also, for these two antigens, the values given for relative sensitivity and relative applied sensitivity are not strictly comparable to those given for the other antigens. As shown in Table I the relative sensitivity values for the plate antigens are less variable than is true for the tube antigens. The ratio of maximum to minimum relative sensitivity for the plate antigens is 140 to 51 or a ratio of 2.74:1 which indicates that the most sensitive plate antigen was 2.74 times that for the least sensitive antigen. For the antigens used in the tube test, the range was from 538 to 36, or a ratio of maximum to minimum of 14.9:1. The relative applied sensitivities for the plate test varied from 187 to 51. In this case the most sensitive

128

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

TABLE I.-Summary of siudies on Brucella antigens used in the Americas and

several European countries I

-

Labo· Coun- ratory try &Antigen

II

III IV V VI VII VIII IX X XI XII

A-1 A-2 B-1 C-1 D-1 E-1 A-1 A-2 A-3 A-4 A-5 A-6 B-1 C-1 C-2 A-1 A-2 B-1 A-1 A-1

A-1 A-1 A-1 A-1 A-1 A-1 A-1 A-2 B-1 C-1 XIII A-1 B-1 B-2 XIV A-1 A-2 XV A-1

Species of Brucella

Type of Antigen

abortus abortus abortus abortus

Plate Tube Plate Plate Plate abortus Plate suis Tube suis Tube abortus Tube abortus Tube meliten. Tube meliten. Tube abortus Plate abortus Plate abortus Tube abortus Plate abortus Tube abortus Tube abortus Plate suis and Plate abortus abortus Tube abortus Tube abortus Plate abortus Tube abortus Plate Tube abortus abortus Special abortus Tube abortus Plate abortus Plate abortus Plate abortus Plate abortus Tube abortus Plate abortus Tubo Plate abortus of Ratio Maximum Minimum

RelaSignifi- tive Acant pied Titer tivity

Cell Conc.

Relative Sensitivity

13.7 0.041 3.1 2.2 12.5 10.3 0.017 0.027 0.017 0.028 0.009 0.024 11.0 14.0 0.140 12.0 0.035 0.028 10.2 4.4

47 86 140 125 65 55 458 467 499 400 538 313 57 55 39 63 86 111 62 136

75 75 75 100

63 115 187 125

6-14-4 14-9-1 22-2-2 18-3-3

50 60 60 60 60 60 60 100 100 100 100 150 100

110 763 778 831 667 897 522 57 55 39 63 86 74 62

24-0-0 24-0-0 24-0-0 24-0-0 24-0-0 24-0-0 4-17-3 5-16-3 5-13-6 2-20-2 9-14-1 10-14-0 6-16-2

0.100 0.037 0.7 0.134 5.2 0.024 20.0 0.080 12.0 7.2 7.0 11.0 ] 0.038 11.0 0.043 7.0

52 130 62 36 126

20

260

24-0-0

75 30 100

82 117 126

12-7-5 14-7-3 17-6-1

100 100

52 51

100 100 100 100

62 79 60 78

52 51 63 62 79 60 78 106

14.9 1

100

Interpre- Readability tation of Test (S*-D*-N*)

3-20-1 11-12-1 5-17-2 9-15-0

Normal Normal Normal Normal Poor Normal Fair Fair Fair Fair Fair Fair Normal Normal Normal Normal Normal Normal Poor Normal Normal Normal Poor Normal Normal Poor Poor Normal Normal Normal Normal Normal Normal Normal Normal

23 1

* S = significant titer, D = doubtful reaction, N = negative reaction. antigen, as far as application is concerned, was 3.67 times the sensitivity of the least sensitive antigen. In comparing the relative applied sensi-

PHYSICAL PROPERTIES OF BRUCELLA ANTIGENS

129

tivity of the tube antigens, the ratio of the most sensitive to the least sensitive was 897 to 39 or a ratio of maximum to minimum of 23:1. TABLE II.-Effect of concentration of Brucella cells and techniques of conducting

the tube agglutination on average titer or relative sensitivity S ecies

Labora-

%Cells CAs by by Vol.

Antigen

A-1 A-3 A-5 C-2 A-2 B-1 A-1

II

III IX

Ratio of

suis abortus melitensis abortus abortus abortus abortus

U. S. B. A. I. Conc. and Technique

Recommended Conc. and Technique

%Cells Cel by Vol.

Sensit i . SRelati*ívie ¡vi

538

Maximum

129

-129 74

14.9

36

Minimum

100 129 74 92 85 77 77

0.045 0.045 0.045 0.045 0.045 0.045 0.045

458 499 538 39 86 111 36

0.017 0.017 0.009 0.140 0.035 0.028 0.134

Relative Sensitivity

1.74

TABLE III.-Agglutination titers on the dried standard Brucella abortus agglutinating serum (International Office of Epizootics) Supplied by the Veterinary Laboratory, Weybridge, England (Tube Test Antigens) Titer on Standard Serum Country

II II

VI IX

XI XII XIII XIV

Laboratory and Antigen

A-2 A-1 A-3 A-5 C-2 A-2 B-1 A-1 A-1 A-1 A-2 B-2 A-2

tivity Values from Table I

Dilution 1:320

1:640

1:1280

1:2560

+ +

+

+ + + +

1 + + +

+ + 1

1 1

+ +3 + + + 3 + + + +

+ + + + + + + 1 + 1 + +

1:5120 1:10240 1:20480

+ + +

1 + +

1 1

86 458 499 538 39 86 111 52 36

1 + 1

79 78

Cell Concentration.-The observations made while conducting the agglutination tests suggested that possibly some of the marked variation in sensitivity found might be due to the particular strain of Brucella used to prepare the antigen and the method of growing the cultures as

130

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

well as harvesting and killing the cells. However, it appeared that the major factors responsible for the great variation in sensitivity were due to variations in the concentration of Brucella cells in the antigens and variations in the techniques of conducting the agglutination test. In order to investigate the latter possibility, seven of the tube antigens which showed a marked range in relative sensitivity were retested using a uniform concentration of Brucella cells and the technic of conducting and reading the test as recommended by the United States Bureau of Animal Industry. The relative sensitivity values obtained by the two methods are shown in Table II. For the recommended concentration and technic of conducting the test given for each of the antigens the relative sensitivities varied from 538 to 36 which is a ratio of maximum to minimum of 14.9 to 1. With a uniform technic of conducting the test and a uniform concentration of cells for all of the antigens the relative sensitivities varied from 129 to 74 or a ratio of maximum to minimum of 1.74:1. These results indicate that the major factors responsible for the marked variation in the results obtained with the tube tests were due, to a very great extent, to variations in the concentration of the Brucella cells in the antigens and to variations in the technic of conducting the test. Results with Standard Dried Serum.-The standard dried seruml of the International Office of Epizootics, kindly supplied by Dr. A. W. Stableforth of the Weybridge Laboratory in England, was used to test 13 of the tube test antigens. The titers obtained on this standard serum are given in Table III. The titers obtained on this one serum were, within experimental error, in general agreement with the relative sensitivity values as given in Table I. DISCUSSION In making the various tests on the antigens involved in this study, particularly the agglutination studies, it is to be expected that the results obtained would not be the same as those which might be obtained in the laboratory which submitted the antigen. Therefore, the values shown should be considered as only rough approximations. With several exceptions, the purity of the antigen from the standpoint of extraneous viable and non-viable bacteria, appeared to be satisfactory for use in the agglutination test. From the standpoint of the character of the agglutination reaction, the sharpness of the end-point of the agglutination titers and the absence of serious discrepancies from the normal with respect to the agglutination titers obtained with the different sera tested, the agglutinability of a high percentage of the antigens appeared to be normal. The studies indicate that uniformity of concentration of the antigen and technic of conducting the agglutination test as well as uniformity in the interpretation of the test would

PHYSICAL PROPERTIES OF BRUCELLA ANTIGENS

131

very materially reduce the wide range of relative applied sensitivities found for the antigens tested. As results of these studies indicate, it is difficult to compare the reports of brucellosis studies which involve serum agglutination tests from different laboratories and countries without satisfactory knowledge of the antigens used, the technics of conducting and interpreting the test. The quite extensive use of the serum agglutination test as a diagnostic test in the bovine brucellosis control program in the United States has demonstrated the importance of a uniform test throughout the country. In the early stages of the bovine brucellosis control program each state had its own particular antigen and technic of conducting the test. Other studies 2 . 3 on a number of such antigens indicated approximately the same marked degree of variation in sensitivity as was found in this study. It was difficult to compare the results obtained in a control program in one state with those of another. Appreciable difficulties also arose with respect to the interstate shipment of cattle which were subject to the serum agglutination test. Many of these difficulties were appreciably alleviated when nearly ten years ago, a uniform antigen and technic of conducting the test was adopted in the United States. ACKNOWLEDGMENT

It is desired to give credit to Dr. Peter J. Ostapchuk for very valuable technical assistance in conducting this study. REFERENCES (1) Stableforth, A. W.: Standardisation du diagnostic de la brucellose bovine par l'épreuve de séro-agglutination. Dix-huitiéme Session du Comité de l'Office International des Epizooties, XXIV: 229-247, (1950). (2) Fitch, C. P. and Thompson, C. M.: Studies of physical properties and agglutinability of Brucella abortus plate antigens from several sources. Cornell Vet., 26: 222-230 (1936). (3) Fitch, C. P., Roepke, M. H. and Thompson, C. M.: Studies of physical properties and agglutinability of Brucella abortus plate antigens from several sources. II. Cornell Vet., 27: 366-373 (1937). ESTUDIOS DE LAS PROPIEDADES FÍSICAS Y LA AGLUTINABILIDAD DE LOS ANTfGENOS DE BRUCELLA USADOS EN LAS AMÉERICAS (Sumario) Para realizar este estudio se utilizó un total de 36 pruebas de aglutinación de antígenos de Brucella, que fueron enviados por 25 laboratorios gubernamentales o particulares de 15 paises distintos. Se hicieron los siguientes estudios: examen microscópico y de cultivo para determinar la pureza de los antígenos; concentración de los antígenos mediante centrifugación para determinar el porcentaje por volumen de células de Brucella; y además, pruebas de aglutinación en 55 sueros de bovinos y caprinos de diversos titulos para determinar la capacidad de aglutinación y de sensibilidad de los antigenos.

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

La pureza de los antígenos, con algunas posibles excepciones, pareció ser satisfactoria desde el punto de vista de su empleo. La concentración de las células de Brucella por volumen en los antígenos empleados para la prueba común de placa, o prueba rápida, varió de 0.70% a 14.0%, con un promedio de 8.6%. La concentración final de los antígenos, según las pruebas de tubo o prueba lenta, varió de 0.009% a 0.14%, con un porcentaje medio de 0.048% de células por volumen. A juzgar por los resultados obtenidos en las pruebas de

los 55 sueros mencionados, un alto porcentaje de los antígenos pareció indicar una capacidad normal de aglutinación. La sensibilidad relativa de los antígenos varió considerablemente entre 36 y 538 según pudo determinarse por los títulos promedios de 24 sueros de título bajo o intermedio. Los estudios efectuados indican que la uniformidad de concentración de los antígenos y la técnica que se siga para las pruebas de aglutinación, podrían reducir muy sustancialmente la variabilidad de sensibilidad relativa encontrada en los antígenos.

FIELD STUDIES ON THE DIAGNOSIS OF ANIMAL BRUCELLOSIS WITH SPECIAL EMPHASIS ON THE RING TEST* By FRED C. DRIVERt AND MARTIN H. ROEPKEt As other papers in this symposium no doubt will deal with the diagnosis of brucellosis in animals other than the bovine, this paper will be confined to the diagnosis of bovine brucellosis and particularly to the use of the ring test for the detection of Brucella infected dairy herds. The extensive use of the blood serum agglutination test for the diagnosis of brucellosis in this country as well as many other countries has, rather satisfactorily, demonstrated the accuracy and practical value of the test under field conditions. The blood test has, however, a handicap which is not related to the accuracy of the test but to the nature of the test, in that it requires a large number of personnel and appreciable funds to use it on an extensive scale in an eradication or control program. It is not practical to conduct county-wide blood tests in counties certified as modified brucellosis-free, at intervals shorter than three years. In such certified counties which adjoin districts not on the area plan, reinfection rates may be quite high, and, in many instances, an interval of three years between blood tests in a county is too long. Many new centers of infection have several years time to spread to neighboring herds. Also, because of the lack of veterinary personnel for the brucellosis control program in our state, we have been able to do very little more than to conduct county-wide blood tests for certification or recertification of the 23 counties which were on the area control plan by 1942. From 1942 to 1949, area control work was initiated in only 5 new counties, which is relatively slow progress considering that there are 87 counties in our state. It was with the thought in mind that the ring test, as an adjunct to the blood test, might provide a means of extending more rapidly or speeding up the control program in our state that a study of the ring test was initiated in the fall of 1947. The ring or ABR test for brucellosis was first described by Fleischhauerl and Fleischhauer and Hermann 2 in 1937 and 1938. The ring test which is applied to whole milk is an agglutination test and depends on the presence of Brucella agglutinins in the milk from an infected animal. The antigen used, a 3 to 4% suspension of Brucella abortus cells, is stained a deep blue color with hematoxylin so that the agglutination reaction may be observed in the presence of whole milk. * Paper No. 2605, Scientific Journal Series, Minnesota Agricultural Experiment Station, University of Minnesota. t Inspector in Charge, U. S. Bureau of Animal Industry, St. Paul, Minnesota.

$ University of Minnesota, St. Paul, Minnesota. 133

134

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

In positive samples, the agglutinated cells adhere to the cream droplets and are carried to the top with the cream to form a deep blue cream ring or line, whereas in milk from non-infected animals the stained antigen remains in the skim milk fraction to give it a light blue color, and the cream ring which forms is white. The nature of the reaction observed gives the test its name. The ring test is very easily performed in that one drop of antigen is added to 1 cc of milk and the mixture allowed to stand at room temperature from 60 to 90 minutes or incubated at 37°C for 45 to 50 minutes which is sufficient time for the cream to rise to the top, at which time the test may be read. The ring test was found to be so sensitive that in a high percentage of cases a positive test was obtained even though the milk from one infected animal was mixed with that from 5 to 15 negative or non-infected cows. This high sensitivity permitted the test to be used on pooled or mixed milk samples from a herd collected at the receiving platform in a dairy plant and thereby serve as a very rapid, and inexpensive screening test to locate infected herds. Since the original description of the test by Fleischhauer, a number of studies have been reported, particularly from Sweden 3 and Denmark.4- 9 These reports described improved methods of preparing the antigen, techniques of conducting the test and rather extensive studies showing the efficiency of the test in locating infected herds. Some idea of the efficiency of the test was presented in reports from Denmark by Seit and Jorgensen 5 and by Christiansen. 9 These investigators found that the ring test was positive on the milk-can samples of 82% of 2,031 herds classified as infected on the basis of the serum agglutination test results. Of the failures of the ring test, investigations of such herds disclosed that about two-thirds of the failures were due to the fact that the infected animals present were not in production. That is, heifers, dry cows or, in some instances, a bull or steer were the infected animals. It was considered likely that the majority of such herds would be detected on a second ring test in three to six months. Such a procedure would overcome, to an appreciable extent, this natural limitation of a single ring test on a herd. STUDIES IN MINNESOTA

The studies in Minnesota are part of a cooperative project between the State Livestock Sanitary Board, the Bureau of Animal Industry of the United States Department of Agriculture and the University of Minnesota. In the early part of the studies the investigations were limited to those counties in which county-wide blood tests were to be made for recertification of the county. The ring tests were made just preceding the blood tests in the county in order that a more accurate evaluation of the agreement between the two tests might be obtained.

DIAGNOSIS OF ANIMAL BRUCELLOSIS

135

The results of the studies in 9 such counties were so encouraging that the ring test work was extended to all of the counties which were on the area plan. In the latter counties, blood tests were made only in those herds which were positive to the ring test. The ring test at six month intervals as an adjunct to the blood test was adopted as a regular part of the control program in the counties under the area plan. At this writing, 25 of the counties have had at least four county-wide ring tests and the fifth test is under way. Methods.-As time does not permit the presentation of the details of the preparation of the antigen, the collection of the milk and cream specimens and the techniques of conducting and reading the test, reference is made for these detailed procedures to previous publications.'° - ' 2 The antigen was prepared from either the official U. S. Bureau of Animal Industry stock Brucella abortus tube or the plate antigen. The antigen was stained with hematoxylin by a slight modification of the method supplied by Dr. Aage Jepsen of the Royal Veterinary College, Copenhagen, Denmark on his visit to our laboratories in 1947. Composite milk samples of approximately 9 or 10 cc were obtained from each herd by collecting about 3 cc quantities of milk from each one of two or three milk cans received from a herd at the receiving platform of the dairy plant or creamery. Cream specimens from each herd were obtained in a similar manner. The ring tests were conducted in a portable laboratory, arranged in a county court house or a public building, or in a trailer laboratory centrally located from the standpoint of the creameries and dairy plants in the area under study. As a fairly high percentage of Minnesota dairymen market cream only, it was necessary to modify the test, to render it applicable to cream. The test on cream was performed by adding 12 drops of cream and one drop of antigen to a small tube containing 1 cc of physiological saline and two drops of a saturated sodium carbonate solution. The sample was then mixed thoroughly, left at room temperature and the results noted as for the test on milk. Arbitrary designations were used to indicate the degree of reaction to the ring test and were given designations ranging from negative to 4+ depending on depth of color of the cream ring as compared with the skim milk fraction. The tests were read as negative if the cream line or ring were lighter in color than the skim milk fraction; 1 + if the blue color of the cream ring were equal to that of the skim milk fraction; 2+ if the color of the cream ring were slightly darker blue than the skim milk fraction; 3+ if the color of cream ring were markedly bluer than the skim milk fraction and 4+ if the color of the cream ring were very dark blue and the skim milk fraction essentially white. Results of Field Studies: Table I is a summary of the study in the nine counties in which the herd ring tests preceded, by a short interval, the

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

136

county-wide blood test. As may be noted from this summary, the overall agreement between the two tests was slightly over 96%. This value by itself is not particularly significant in that the average percentage of infected herds in these counties was approximately 4.5%. If all of the ring tests had been negative there would have been approximately 96.5% agreement. Of major interest are the values shown in the right hand column which indicate the per cent efficiency of the ring test in locating the infected herds or the per cent of infected herds which were positive to the ring test. An average of 68% of the infected herds were positive to the test. Of the 120 herds which were negative to the test, 107 were investigated to determine the possible cause or causes for the failures. Of the 107 failures to detect the infected herds, 64% were due to non-producing reactors present. Usually only one such animal was TABLE I.-Comparison of results of blood tests with herd ring tests in counties with county-wide blood tests County

Koochiching Itasca St. Louis

No. Herds Ring Tested

228 584 2,704

Agreement of Ring and Blood Tests BloodRing -

210 573 2,502

Blood + Ring +

Per cent Agreem't

11 4 61

96.0 98.8 94.7

Disagreement Blood + Ring -

Blood Ring +

Per cent Efficiency in Locating Inf. Herds

5 2 20

2 5 121

69 67 75

49

48

1

100.0

0

0

100

Watonwan Wilkin Hubbard Kitson Roseau

828 669 1,054 990 1,363

748 558 1,014 906 1,326

56 69 15 33 14

97.8 94.8 98.5 94.9 98.1

15 15 15 39 10

9 26 10 12 13

79 82 50 46 58

Total

8,469

7,885

264

96.2

121

198

Lake

1

68

present per herd. The ring test was positive on approximately 90% of the infected herds in which one or more infected animals were in production at the time of the test. In the last three counties listed in Table I, the ring tests were conducted during the late summer or early fall months during which time an appreciable percentage of the dairy cows were dry. This suggests that to obtain best results the ring tests should be made during the season of highest milk production in an area. The number of herds positive to the ring test but negative to the blood test (false positive ring test) was unusually high in St. Louis County. This was due to a deliberate disregard of the warning of the Danish workers with respect to the collection of milk samples from the weighing tank on the receiving line in the dairy plants. The ring test is so sensitive that the small amount of milk from an infected herd which remains

DIAGNOSIS OF ANIMAL BRUCELLOSIS

137

in the weighing tank is sufficient, in many instances, to cause a positive reaction on the sample from a negative herd which follows. The results of blood tests in 650 herds positive to the ring test on cream and 650 herds positive to the ring test on milk are shown in Table II and Figure 1. An over-all agreement of the two tests of 70% for the positive ring test on cream specimens and 62% for the positive ring test on milk was found. It was considered that there was agreement between the two tests even though some herds were classified as suspicious on the basis of the blood test results. As might be expected, the percentage of herds classified as reactor herds was highest for those on which 3 or 4+ ring tests reactions were obtained. TABLE II.-Results of blood tests on herds positive to ring tests Positive Herd Cream Tests Comparisons

120 + Tests R* SI NI

197 ++ R

S

226 +++ N

R

S

98 ++++ N

R

Number of herds... 38 13 78 115 25 57 159 20 47 77 Per cent of herds... 29 10 60 58 13 29 70 9 21 79

S

N

Agreement

R

S

Tota No. Herds

N

7 14 385 65 96 650 7 14 60 10 30 's, 70 &

Positive Herd Milk Tests 88 + Tests

145 ++

150 +++ --

267 ++++

Number of herds... 10 9 69 42 18 85 79 23 48 193 28 46 324 78248 650 Per cent of herds... 11 10 78 29 12 59 53 15 32 72 11 17 12 38 ~, 62

R = Herds with one or more animals positive to blood test. S = Herds with one or more animals giving "suspect" titers but no reactors. N = Herds negative to blood test.

As may be noted from Figure 1, it would appear that the test as used on the cream specimens was less sensitive than that for milk. Another point of interest is that the percentage of herds classified as suspicious on the basis of the blood test results was relatively constant irrespective of the degree of reaction to the ring test either on milk or cream. Table III is a summary of the results obtained in those counties in which county-wide blood tests were made either between the second and third or between the third and fourth ring tests. The first two counties are predominately dairy counties whereas the other three counties contain a fairly high percentage of beef herds. Carlton County is on the border of a district which is not on the area control plan and has had a history of a high reinfection rate between county-wide blood test. The third ring test was made shortly after the county-wide blood test was started. The county-wide blood test was conducted by the

138

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

local veterinarians and a period of approximately 9 months was required to complete the work. It is reasonable to assume that some of the 17 infected herds disclosed first by the blood test became infected in the interval between the two tests. This is suggested by the fact that the fourth ring test which was made within two weeks after the completion of the county-wide blood test disclosed 12 new infected herds contained 18 reactor animals. Of the 17 infected herds which were first disclosed by the county-wide blood test, 3 were positive to the fourth ring test in the county. It is of considerable interest to note that the three ring tests plus the county-wide blood test disclosed a total of 97 Numbers on top of. blocts - Total numberherds in eochgroup

l[-

Herds Cassed os Negotive on Blood Test D* 'iuect» ' ..

_

88

. 145 197

129

...

t.nfected" .

150 226

"

267 98

6 4

2

·--

+-I+t'+ Rino Test Reodinos on Herds

+++'++

FIGURE 1.-Results of blood tests on herds showing various degrees of reactions to the ring tests

new infected or reactor herds, whereas the four ring tests alone would have disclosed at least 95 new reactor herds. The results obtained in Clearwater County, also a predominately dairy county, were very similar to those obtained in Carlton County. Of the four infected herds which were first disclosed by the county-wide blood test, 3 were positive to the third ring test in the county along with one additional new infected herd. Although there was a time interval of approximately four months between the completion of the county-wide blood test and the third ring test, the three county-wide ring tests disclosed as many new centers of infection as were found by two countywide ring tests plus the county-wide blood test.

DIAGNOSIS OF ANIMAL BRUCELLOSIS

139

If the relatively high percentage of beef herds in the other three counties listed in Table III are taken into consideration, the results obtained with the ring test in these counties would appear to be reasonably satisfactory. It is evident, however, that if a ring test program were to be adopted as an adjunct to the blood test for brucellosis control in TABLE III.-Results of county-wide blood tests following two or three county-wide ring tests for brucellosis Ring Tests New Infection County

No.s Herds

No. Test

New Inf. Herds

Carlton Clearwater Clay Marshall Norman

1601

1302

1506

2084

1588

New Inf. Cattle

New Inf. Herds

3 4th

80 12

365 18

(13, + ring

2 3rd

12 1

52 2

3, + + ring ring

3 4th

54

222 1

3 4th

65

2 3rd

39

1

9

10

%of Total New Infection Disclosed by Ring Test

County Blood Test Additional Infection

288 23

(20

233 24

48

New

Inf.

Herds

Cattle

19

82%

95%

5

75%

91%

41

75%

85%

45

76%

85%

92

45%

72%

Cattle

1, + ring

TABLE IV.-Partial county blood tests for recertification following three county wide ring tests in Pennington and Red Lake counties Pennington (Est. 1000 hds., 17,000 cat.) New inf. disclosed No. Herds

Three ring tests ........................ Blood tests (491 herds, 6926 cattle) ...... (243 herds, 4262 cattle) .....

18 3

No. Cattle

59 3

Red Lake (775 hds., 15,000 cat.) New inf. disclosed No. Herds

No. Cattle

36

156

2

2

Blood tests were conducted on only old reactor herds plus those herds which had not had one or more ring test.

areas of this nature, provisions should be made for blood tests at suitable intervals on all herds which are not covered by the regular ring test program. Table IV is a summary of the results obtained in two counties, also containing an appreciable percentage of beef herds, in which tests were made only in those herds which had not had one or more ring tests

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plus those herds which were on the infected list; The results in these two counties suggest that the major portion of the infection was confined to the herds producing market milk or cream. Relatively little infection was found in the large number of herds in these counties from which cream or milk samples had not been obtained for the ring test. CONCLUSIONS The fact that the ring test was negative on approximately one-third of the infected herds and from the fact that the blood tests were negative on approximately one-third of the herds which were positive to the ring test, the ring test cannot be considered a highly reliable test on a pooled milk or cream sample from a herd. It would seem more logical to consider the test as a highly sensitive presumptive or screening type of test which, to be effective, should be used in conjunction with the blood test in an official disease control program where there are provisions for follow-up blood tests in all herds which are positive to the ring test and where there are provisions to give assistance and counsel to the owners of the infected herd disclosed. It is a test that may be used to direct the blood testing work to those herds most likely to be infected, which provides a rapid and inexpensive means in conjunction with the blood test of locating infected herds or new centers of infection. Used in this manner, it may offer a means of appreciably speeding up the brucellosis control program in many areas where dairying is the predominate cattle industry without an appreciable increase in cost and in personnel, both of which are major factors at this time in a brucellosis control program. The inaccuracies and natural limitations of the test may be overcome to a great extent by repeating the test at six month intervals. The effectiveness when used in a program of this nature is indicated by the results in the two predominately dairy counties, namely Carlton and Clearwater. The number of new centers of infection which were disclosed by the county-wide blood test alone was very low. The results obtained in these two counties along with the evidence obtained from preliminary summaries of the results obtained in several other similar counties indicate to us that there is a rather limited justification for conducting county-wide blood tests for recertification of counties of this nature when the limited personnel we have and the funds involved could be used much more advantageously and effectively to extend the control and eradication program to new counties and districts. We are convinced that a control program of county-wide ring tests alone at six month intervals will keep the number of unknown centers of infection in a predominately dairy county at an appreciably lower level than would be obtained with county-wide blood tests alone at intervals of 3 years. The over-all requirements in terms of personnel and funds would also be appreciably lower.

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The relatively high percentage of false positive ring tests found in this study, although objectionable, does not seriously interfere with the use of the test as an adjunct to the blood test in counties of the type involved in this study. The average per cent of infected herds in the counties was below 2.5%. The problem of the false positive ring test is somewhat similar to that of the "suspect herd" found with the blood test, that is, the herd with one or two animals with suspicious or doubtful titers and all other animals negative to the blood test. We have frequently experienced with county-wide blood tests nearly as many, and in some instances more, suspect herds than herds classified as infected on the basis of the blood test results. Our data indicate an increased percentage of false positive ring tests with the successive tests in the counties. There would appear to be a low base line of false positive tests in counties in which there has been relatively little infection for a number of years. Another factor which contributes to the number of false positive ring tests is the tendency for a positive test on a herd to persist for a period of time after the animals designated as infected by the blood test are removed from the herd. The response of the herd owners to the ring test program has been excellent. In fact, the response might even be termed "enthusiastic". In the past the herds have gone from three to six years without a blood test. The dairymen become concerned regarding the status of their herds during these long intervals. The frequent checking of the herds provided by the ring test, is one of the chief reasons for the enthusiasm of the dairymen to the test. From the disease control standpoint, it is very desirable to locate new centers of infection as early as possible in order to reduce the spread of the disease both in the herd as well as to neighboring herds. In this respect, it has been of considerable interest to us to note on the successive ring tests in a county that an increased percentage of the new infected herds contained but one or two reactors. Early disclosure of an infection is of major importance to the herd owner. Very limited studies have been made on the effect of vaccination, with Brucella abortus Strain 19, on the test. A study of several adult animals which were followed closely after vaccination, along with the experiences obtained with the test in the field, suggest that vaccination with Strain 19 may interfere to some extent with the ring test as a diagnostic aid. Three adult animals vaccinated with Strain 19, although strongly positive to the ring test shortly after vaccination, became negative to the test in 4 or 5 months, whereas the blood titer remained appreciably above 1 to 100. These results along with the experiences of the test under field conditions suggest that the majority of animals with a persistent blood titer due to vaccination, are negative to the ring test. These limited observations are in agreement with the report of Traum and Maderious1 3 on milk-whey agglutination titers of animals with

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vaccinal titers as compared with those from which virulent Brucella had been isolated from the milk. Of 50 adult animals vaccinated with Strain 19, 48 became negative to the whey agglutination test at the 1:12.5 dilution within 3 months after vaccination although the animals remained positive blood reactors. Of 89 animals from which virulent Brucella had been isolated from the milk in 85 cases, the highest whey titers for the milk from any one of the 4 quarters were above 1:12.5. Similar observations were reported by Holm, Eveleth and Rheault with the ring test on milk from animals with persistent vaccinal titers when such milk was diluted to varying degrees with negative milk. They found in a high percentage of cases that the ring test on such milk at dilutions above 1:10 with negative milk, was negative whereas the opposite was true with milk from animals with positive blood reactions and a history of long-standing infection. The estimated cost of a county-wide ring test is about 10% of that of a county-wide blood test in areas where the infection rate is low, as was true in this study. However, the cost would increase appreciably as the infection rate increased due to the cost of conducting the necessary follow-up blood test on the herds which were positive to the ring test. The reports by Bruhn and by Christiansen on the results of simultaneous blood and ring tests on individual animals suggests that the ring test is of limited value on an individual animal basis. They found a significant percentage of positive ring tests on animals which were negative to the blood test. ACKNOWLEDGEMENTS

It is desired to give credit for the large amount of field work involved in this cooperative study to members of staff of the U. S. Department of Agriculture, Bureau of Animal Industry, namely, Doctors L. B. Calusen, J. E. Wentworth and Louis Olson. REFERENCES (1) Fleischhauer, G.: Die Abortus-Band-Ringprobe (ABR) zue Festellung von bangverdachtigen Vollmilchproben. Berl. tierarztl. Wchnschr., 53: (1937): 527-528. (2) Fleischhauer, G., and Hermann, G.: Ueber weiters Erfahrungen mit der Abortus-Bang-Ringprobe (ABR) bei der Untersuchung Von Milchproben auf Abortus Bang. Berl. tierarztl. Wchnschr., 54: (1938): 333334. (3) Norell, N., and Olsen, A.: Om várdet av serologiska mjolkundersoknigar medlst "ABR" (Abortus-Bang-Ringprobe). Skand. Vet.-tidskr., 38: (1943): 321-341. (4) Winther, O., and Hansen, A. C.: Undersgelser over Abortus-Bang-Ringproven. Maanedsskr. Dyrl., 55: (1943): 401-416. (5) Seit, B., and Leth Jergensen, K.: Abortus-Bang Ringprovens Vaerdi som diagnostisk Hjaelpemiddel. Maanedsskr. Dyrl., 56: (1944): 227-292. (6) Bruhn, P. A.: Abortus-Bang-Ringproven Valleog Serum-titer. Medlemsbl. Danske Dyrl., 27: (1944): 477-502.

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(7) Bruhn, P. A.: Problemer der Knytter sig til Abortus Bang-Ringproven. Skand. Vet.-tidskr., 38: (1948): 71-93. (8) Bruhn, P. A.: The Brucella Abortus Ring Test. Am. Jour. Vet. Res., 9, (1948): 360-369. (9) Christiansen, M. J.: Om Abortus-Bang-Ringproven og dens Anvendelse i den praktiske Kastningsbekaempelse. Maanedsskr. Dyrl., 69: (1948): 193-230. (10) Roepke, Martin H.; Clausen, Lawrence B.; and Walsh, Andrew L.: The milk and cream ring test for brucellosis. Proc. 52nd Ann. Meeting, U. S. Livestock Sanitary A., (1949): 147-159. (11) Roepke, Martin H.; Paterson, Katherine G.; Driver, Fred C.; Clausen, L. B.; Olson, Louis; and Wentworth, J. E.: The Brucella abortus ring test. Am. Jour. Vet. Res., XI (1950): 199-205. (12) Roepke, Martin H.: The Brucella abortus ring test. Brucellosis (A Symposium) by the Am. Assoc. Adv. Sci., Washington, D. C., 1950, pp. 126135. (13) Traum, J., and Maderious, W. E.: The interpretation of whey agglutination test results in cows vaccinated with Brucella abortus strain 19. Am. Jour. Vet. Res., 8 (1947): 244-246. DIAGNOSTICO DE LA BRUCELOSIS EN LOS ANIMALES, CON REFERENCIA ESPECIAL A LA PRUEBA DEL ANILLO (Sumario) De conformidad con el plan para el control de la brucelosis en toda una zona, en 28 condados se ejecutaron de tres a cuatro pruebas del anillo en muestras de leche y de crema. Al iniciarse los estudios menos de 5% de los rebaños estaban infectados. En nueve condados se hicieron pruebas sanguíneas de todo el ganado después de las pruebas del anillo. En otros seis condados las pruebas sanguineas se hicieron después de dos o tres pruebas del anillo. En estas condiciones (menos de 5% de rebaños infectados) se obtuvieron los siguientes datos del estudio comparado: cuando s61o se practicó una prueba del anillo, esta resultó positiva en mezclas de leche y crema del 68% de los rebaños infectados. Aproximadamente dos terceras partes de los fracasos de la prueba del anillo se debieron a que los animales infectados (por lo común sólo uno) no se encontraban en producción al hacer la prueba del anillo. Esos rebaños infectados se descrubren por lo general al realizar una prueba del anillo en todo el condado seis meses después, cuando los animales infectados están en producción. La prueba resultó positiva aproximadamente en 9 de 10 rebaños con uno o más animales infectados en producción. En los condados en que predomina la producción de leche, 3 6 4 pruebas del anillo en todo el ganado, realizadas con intervalos de 6 meses, revelaron aproximadamente 85% de los rebaños infectados y 95% del ganado infectado. En varios de los condados en que se hicieron pruebas de sangre entre la tercera y cuarta pruebas del anillo, se descubrió que las cuatro pruebas del anillo revelaron prácticamente el mismo número total de nuevos centros de infección que las tres pruebas del anillo más la prueba sanguínea en todo el condado. De los rebaños positivos a la prueba del anillo, aproximadamente la tercera parte resultaron negativos con la prueba sanguinea. Las discrepancias o errores de esta naturaleza son algo parecidos a las discrepancias observadas con la prueba sanguínea, en que muchos rebaños se clasifican como sospechosos debido

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a la presencia de uno o dos animales con un título sanguíneo sospechoso. Tomando por base los resultados de las pruebas sanguíneas posteriores, se clasifica como negativo un elevado por ciento de estos rebaños. Aunque ofrece reparos, el problema de las pruebas del anillo aparentements seudopositivas no afecta en forma marcada el empleo de la prueba como presuntiva para los rebaños. Como prueba presuntiva permite limitar las pruebas sanguíneas a los rebaños que es más probable estén infectados, permitiendo así evitarla en numerosos rebaños negativos o no infectados. Los estudios limitados en la leche de animales adultos vacunados con la Cepa 19 de Brucella abortus indican que en un por ciento relativamente elevado de casos a los 4 a 6 meses de la vacunación la leche de dichos animales es negativa a la prueba del anillo, o negativa a diluciones relativamente bajas con leche de animales no infectados.

COMPARATIVE STUDY OF MEDIA ORDINARILY USED FOR THE GROWTH OF BRUCELLA SPECIES By GENESIO PACHECO AND MILTON THIAGO DE MELLO The need for a liquid medium yielding good and early growth of Brucella species, stimulated a large amount of research in various laboratories. Tryptose broth and trypticase broth are at present the most widely used as were some time ago liver broth and placenta broth. Chemically defined media, although of great advantage for chemical studies of Brucellae are not well suited for rapid and simple production of large quantities of microorganisms. In order to reexamine this question, experiments were carried out with some of the media ordinarily used for the growth of Brucellae. Strains of the three Brucella species were grown in meat extract broth, veal infusion broth, dextrose veal infusion broth, liver citrate broth, bovine placenta broth and placenta glycerol broth. The intensity of growth was estimated with the aid of "Lumetron" (Mod. 400-G) interposing filter No. 530. The media were dispensed in test tubes in amounts of 10 ml and were inoculated with 0.1 ml of a 48 hour culture of each of 10 strains of Brucellae from our culture collection. After 48 hours of incubation at 37°C, the cultures were transferred to matched test tubes and the optical density was measured. We observed the following decreasing graduation in the ability of the media to support the growth of Brucellae: placenta broth, meat extract, dextrose veal infusion broth, meat infusion broth, glycerol placenta broth and citrate liver infusion broth. Similar experiments were conducted using veal infusion broth, dextrose veal infusion broth, liver infusion broth, placenta infusion broth and tryptose broth, but nine strains of Brucellae were used and the intensity of growth was measured by the same method after 20 hours. The best results were observed with veal infusion broth followed in decreasing order by placenta infusion broth, tryptose broth, dextrose veal infusion broth, and liver infusion broth. In both experiments liver infusion broth yielded the poorest growth. The preliminary experiments summarized above showed that Brucellae could be grown in relatively simple media and that veal infusion broth seemed better than tryptose broth for the early development and this fact could be seen even by visual comparison. The reading of the optical density of cultures in the "Lumetron" in low concentrations of microorganisms was not very sharp and this fact led us to more detailed work using greater number of media and measuring the intensity of growth with the Pulfrich nephelometer. All the results presented in this paper represent the differences between the initial 145

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relative turbidity of the media and the relative turbidity of the same media inoculated and after a variable time for each experiment. The relative turbidity was the mean of 5 readings in the apparatus using filter No. 1 (red) and standard turbidity disc No. 2. In all the experiments we used the strain 955 of Brucella suis received from "Instituto Biológico de Sao Paulo" and originally from Dr. Huddleson. The media were dispensed in standard 16 x 160 mm test tubes (Pyrex) in amounts of 10 ml unless otherwise stated and were inoculated with 0.1 ml of a 20-24 hour culture of microorganisms generally in tryptose broth. 1. In preliminary experiments we used media prepared by two types of pancreatic digestion (A and B) of bovine placenta, beef heart and bovine spleen and liver, dextrose broths prepared with the same organs, veal infusion and dextrose tryptose broth. Digested medium A was prepared according to the technique of Brown,* with 1% dextrose; in some batches a turbidity was observed after the sterilization and the medium was again filtered and sterilized the same way (115°C-20 minutes); the pH changed from 6.8-7.2 to 6.7-7.0; both were used in the tests. Digested medium B was being employed in this laboratory for production of gas gangrene toxins and was prepared as follows: to each 500 gm of ground fresh organs, 1000 ml of distilled water were added and the mixture was heated at flowing steam during 40 minutes; the reaction was adjusted to about pH 8.4 and 6 gmr of pancreatin were added. The mixture was digested at 500C overnight; in order to avoid contaminations some batches were digested under toluene; the following day the medium was filtered through cotton gauze and the pH adjusted 4.2; the medium was kept in the refrigerator at 5°C overnight; the clear supernatant was filtered through paper and the pH adjusted to 7.6; 1% peptone (Bacto), 1% dextrose (Pfanstiehl) and 0.5 sodium chloride were added. The medium was heated during 10 minutes at 115°C, filtered through paper while hot, distributed and sterilized (115°C for 20 minutes). Dextrose broth (1% peptone, 1% dextrose, 0.5% sodium chloride) was prepared by the conventional methods. Spleen infusion broth presented a high final pH (9.2) and was deep brown; it was adjusted to about pH 7.4 and cleared by mixing it with equal parts of a phosphate buffer solution. Liver infusion broth was cleared with equal parts of distilled water. The pH of all media before the final sterilization ranged from 7.4 to 7.9 but almost always dropped to very low figures after that, as seen in Table 1. Dextrose tryptose broth (1% dextrose) had a final pH of 7.2. Veal infusion broth was obtained from the stock of general media room of the Institute. * Brown, J. H.: Media prepared by the pancreatic digestion of meat. Jour. Bact., 1948, 55 (6): 817-872.

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In that first experiment turbidities were read in the nephelometer with different color filters and standard turbidity discs and as the results were almost the same, we will present only the data obtained with filter No. 1 (red) and disc No. 2, which were the same used in subsequent experiments. The results after 48 hours of incubation at 37°C are shown in Table 1. The results of this preliminary experiment indicated that the best media for the growth of Brucella suis strain 955 in 48 hours were the following, in decreasing gradation: (1) beef heart digested medium A; TABLE

1.-Difference between relative turbidity before and after 48 hour incubation of different media inoculated with Br. suis Medium

pH

Difference

6.8 6.8 6.5 6.5 6.1 6.5 6.1

161.8 106.1 82.3 71.4 52.6 42.2 17.0

6.7 6.5 6.4 6.6 6.5

53.8 7.1 71.7 0.5 22.4

6.3 6.5 6.5 7.8

81.6 147.7 8.3 0.7

7.0 6.5 6....

72.0 44.0

Digested medium A

Beef heart ............................... Beef heart (sterilized twice) .............. Bovine placenta (ster. twice) ............. Bovine liver ............................. Bovine liver (sterilized twice) ............ Bovine spleen ............................ Bovine spleen (sterilized twice) ........... Digested mediumz B

Beef heart (digested without toluene).... Beef heart (digested under toluene) ...... Bovine placenta (dig. under toluene)..... Bovine liver (dig. under toluene) ......... Bovine spleen (dig. under toluene) ....... Dextrose broth

Beef heart .............................. Bovine placenta .......................... Bovine liver ............................. Bovine spleen ............................ Controls

Veal infusion broth ...................... Dextrose tryptose broth .............

(2) dextrose placenta broth; (3) beef heart digested medium A sterilized twice; (4) bovine placenta digested medium A sterilized twice; (5) dextrose beef heart; (6) veal infusion broth; (7) placenta digested medium B under toluene, and (8) liver digested medium A. Some media yielded a fair growth and other a poor growth as some spleen and liver media. This fact was possibly due to repeated filtrations and sterilizations as well as variations in their final pH. 2. In another experiment the media were buffered during their processing and to part of them 1% peptone were added in order to see whether the growth could be improved. The media were prepared by pancreatic digestion following Brown technique and after filtration the

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volumes were completed to 1,000 with distilled water and the pH adjusted to 7.2. To each liter, 5.76 gm Na2 HPO 4 and 0.48 gm KH 2P04 were added; the media were heated in flowing steam for 10 minutes, filtered while hot and kept in the refrigerator overnight. To one part of the media 1% dextrose and 0.5 sodium chloride were added; to the other part the same substance and 1% peptone were added. They were heated again in flowing steam for 10 minutes, filtered, dispensed in flasks and sterilized at 115°C for 30 minutes. The slight precipitate formed was avoided by pippeting 10 ml of the supernatant to the tubes for the tests. Dextrose tryptose broth contained 2% tryptose, 1% dextrose and 0.5 sodium chloride. After inoculation the tubes were incubated and the turbidity read after 24 hours. TABLE 2.-Difference between relative turbidity before and after 24 hour incubation of different digested media buffered at pH 7.2 with and without peptone, inoculated with Br. suis Medium

Digested medium A Beef heart ........................................ Beef heart, with peptone ......................... Bovine liver ..................................... Bovine liver, with peptone ........................ Bovine placenta .................................. Bovine placenta, with peptone ................... Bovine spleen .................................... Bovine spleen, with peptone ...................... Controls Dextrose tryptose broth .......................... Veal infusion broth ...............................

Difference

5.5 0.4 1.1 0.2 4.8 0.6 0.5 0.2 17.5 21.1

The results are shown in Table 2. The results indicate that the buffering of the media does not improve the growth of the strain tested and was even unfavorable for that purpose, while in dextrose tryptose broth and veal infusion broth the growth was fairly good. Peptone did not improve the growth and some media, as beef heart, gave better results without peptone. 3. A new series of tests was conducted preparing media only with beef heart and following all the methods referred above. Placenta media were not prepared because they gave rise to difficulties during filtration. The results are shown in Table 3. Again in that experiment the controls supported better growth than the digested media; only in digested medium A good early growth was observed, which became better than that observed in dextrose tryptose broth in 48 hours.

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4. In order to verify whether beef heart digested medium A contained growth promoting substances which could be extracted from it and possibly improve the growth of Brucellae when added to other media, two parallel experiments were conducted. Beef heart digested medium A was mixed with ether (1:1) and kept undisturbed in a separatory funnel overnight; the medium in the botTABLE 3.-Difference between relative turbidity before and after 24 and 48 hour incubation of media preparedwith beef heart, inoculated with Br. suis Differences Medium 24 hrs.

48 hrs.

medium A, buffered ............. medium A ...................... medium A (sterilized twice) ... medium B, buffered ............. medium B ...................... broth ...........................

8.9 17.6 9.0 0.7 1.7 9.3

27.9 35.3 24.3 1.0 3.4 22.3

Controls Dextrose tryptose broth .............. Veal infusion broth ...................

19.6 19.8

30.4 45.1

Digested Digested Digested Digested Digested Dextrose

TABLE 4.-Difference between relative turbidity before and after 20 and 42 hour incubation of media with extracts of beef heart digested medium A, inoculated with Br. suis Differences Medium

Dextrose tryptose broth + ether - extract........... Veal infusion broth + ether - extract .............. Dextr. tryp. broth + acetone - extract ............. Veal infusion broth + acetone - extract ............ Digested medium A ether-extracted ................. Digested medium A acetone-extracted ............... Controls Digested medium A ............................. Dextrose tryptose broth ......................... Veal infusion broth .............................

20 hrs.

42 hrs.

16.4 17.8 10.8 14.8 4.8 2.9

24.3 36.8 16.4 28.5 11.7 9.6

4.4 13.6 14.1

10.6 24.4 28.5

tom was dispensed in the test tubes in amounts of 10.5 ml and the supernatant ether solution was added to dextrose tryptose broth and veal infusion broth (5 ml of ether solution and 10 ml of media). Another part of the digested medium was mixed with acetone (1:1), kept in the refrigerator overnight, and centrifuged. The acetone in the supernatant was evaporated in front of a fan and dispensed in the tests tubes in amounts of 12 ml. The precipitate was washed twice in acetone,

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centrifuged and partially dried; the residue was diluted in distilled water and 0.5 ml of the dilution was added to tryptose broth or to veal infusion broth. The media were sterilized at 115°C for 20 minutes, as well as the controls (medium without extraction, tryptose broth and veal infusion broth). The readings of the turbidity were made after 20 and 42 hours and the differences of turbidity are shown in Table 4. The experiment showed that veal infusion broth plus ether-extract of digested medium A was better than the other in 20 and 42 hours. Dextrose tryptose broth plus the same extract was better than the control in 20 hours and almost equal in 42 hours. CONCLUSIONS The experiments showed that very elaborated media are not necessary for the growth of Brucella and that the common media dextrose tryptose broth and veal infusion broth are adequate for that purpose, at least when stock cultures are being cultivated. For isolation we used any of the two referred media plus 5% horse serum. Some batches of tryptose were markedly bacteriostatic when the medium did not contain dextrose or horse serum. ESTUDIO COMPARATIVO DE LOS MEDIOS USADOS COMtNMENTE EN EL CULTIVO DE ESPECIES DE BRUCELLA (Sumario) Con el objeto de seleccionar los mejores medios comunes para el cultivo de especies de Brucella, realizamos experimentos preliminares de diez cepas de las tres especies de Brucella cultivadas en los siguientes caldos: infusión de res, extracto de carne, infusion de higado citrado, infusion de carne glucosa (dextrosa), infusion de placenta bovina y triptosa. Se midió la intensidad de crecimiento después de 24 horas de incubación a 37°C, con un colorímetro fotoeléctrico "Lumetron". En estos experimentos se comprobó que el mejor medio es la simple infusión de caldo de res, y después, la infusión de caldo de placenta bovina. Después de 48 horas de incubación, el crecimiento fué ligeramente más abundante en caldo de placenta bovina que en la simple infusión de caldo de res. Luego se realizó otra serie de experimentos con una cepa de Brucella suis cultivada en varios medios liquidos. La intensidad de crecimiento fué medida con un nelfelómetro (Photometre de Pulfrich pur la mesure des troubles). Los caldos usados fueron los mismos empleados en los experimentos preliminares, y además se utilizaron varios tipos de digestos trípticos de carne de ternera, músculo de corazón de res, hígado, bazo y placenta. Algunos de los medios fueron también tamponados al pH 7.2. La simple infusión de caldo de res produjo un crecimiento más abundante que los otros medios ensayados. En unos pocos casos, algunos medios mostraron notable resistencia a la Brucella suis, incluyendo algunos lotes de caldo de triptosa.

THE CARBON DIOXIDE REQUIREMENTS OF BRUCELLA ABORTUS

By A. G. MARR AND J. B. WILSON Research Assistant and Associate Professor, respectively, Department of Agricultural Bacteriology, the University of Wisconsin, Madison, Wisconsin A rational approach to chemotherapy of brucellosis requires that we have among other things an understanding of the physiology of the Brucellae. The establishment during the past fifteen years of the theory of competitive inhibition of enzyme systems in microorganisms of chemical agents has opened a new approach to the problem of chemotherapy. The literature on this subject and its application to treatment of disease has been clearly presented in the monograph, "The Basis of Chemotherapy" by Thomas and Elizabeth Work. This means, then, that we are in need of information on the mechanism of synthetic and of energy yielding reactions as carried out by the Brucellae. When we have this information we may be in a much better position to predict what agents might be used in chemotherapy of brucellosis. We must keep in mind, however, that the enzymatic reaction we wish to block in the bacterium should be unique to it and not be a reaction that would be blocked equally as well in the host. A study of the carbon dioxide requirements of Brucella abortus is indicated here for it is well known that cases yield cultures that require added carbon dioxide for primary isolation. Specific blocking of the mechanism for assimilation of carbon dioxide is a likely step in chemotherapy of Brucella abortus infections, since the metabolism of carbon dioxide in the parasite may differ from that of the host. It was recognized very early that Br. abortus was difficult to cultivate in air, but that growth could be obtained by using sealed culture tubes. Bang, in 1897, using different atmospheres of O2 and CO2 thought that growth of the organism was influenced by the lowered pO2. Novak, in 1908, cultivated Brucella abortus on the surface of solid media by enclosing the tubes in a jar with actively growing cultures of the highly aerobic Bacillus subtilis. Novak, as Bang had done previously, interpreted his data in terms of the lowered pO2 brought about by the growth of Bacillus subtilis. Numerous other workers reported growth of other pathogens such as the gonococcus and the meningococcus using Novak's technique, and their data were interpreted to mean these organisms required a lowered PO2. There were, however, some dissenters from this view and notable among them was Gates who, in 1919, thought that the change in pH brought about by the increased pCO2, might be a determining factor in the growth of the meningococcus. In 1921, Huddleson 151

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showed that chemically generated C02 introduced into the culture environment in a concentration greater than that of air would stimulate the growth of Brucella abortus and that the growth was not due to a reduced pO2. Huddleson concluded that a concentration of 10% C02 in the atmosphere was optimum for the growth of the abortion organism and that this pCO2 made the pH of the medium more optimal for growth. Next, Theobald Smith demonstrated that the size of the inoculum influenced the pCO2 required for growth; the larger the inoculum the less CO2 required. Smith showed also that C02 could not be replaced with hydrogen or nitrogen and he expressed doubts that the presence of C02 simply adjust the pH of the medium to that favorable for Brucella abortus to grow. Finally, in 1931, G. S. Wilson proved that C02 per se is required and that bicarbonate would not substitute for it. Wilson concluded that C02 acts not by altering the acidity of the medium but by virtue of its power of diffusing rapidly through the intact cell wall, and giving rise to an increase in the intracellular hydrogen-ion concentration. Let us look for a moment at the C02 requirements of other heterotrophic microorganisms. Valley and Rettger as well as Gladstone, Fildes, and Richardson have pointed out that the requirements for carbon dioxide among heterotrophic bacteria is extremely widespread. Apparently the requirement of Brucella abortus for a pCO2 greater than that of air is unique only in a quantitative sense. C02 is known to play an important role in heterotrophic metabolism for Wood and Werkman demonstrated ten years ago the fixation of CO2 by a heterotrophic bacterium and, more recently, Ajl and Werkman have shown C02 to be necessary in the cellular synthesis of dicarboxylic acids. Pappenheimer and Hattle and, also, Buchanan, Sonne, and Deluva have shown C02 to be necessary in the synthesis of nucleic acid constituents. Lwoff and Monod have indicated that C02 is probably involved in the synthesis of other, as yet unknown, intracellular catalysts. As a part of our investigations into the metabolism of the Brucellae, and in particular the C02 metabolism, the stability of Br. abortus for added carbon dioxide was determined. It is well known that strains of Br. abortus which require added C02 for growth can be trained to dispense with their added CO2 requirement. When slants are heavily inoculated with carbon dioxide requiring strains and incubated in air, usually one or more colonies develop on the slant. Theobald Smith interpreted this to mean that the single bacterium had been kept alive by the C02 production of many perishing bacteria. However, if these few colonies are tested it is found that they have lost the requirement for added carbon dioxide. This suggested to us a mutational loss of the added C02 requirement. For the determination of mutation rate, flasks containing 100 ml of Albini brucella broth were inoculated with 104 to 105 cells from a 24 hour

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culture grown under 10% carbon dioxide. One ml of the inoculated broth was then transferred aseptically to each of 15 sterile (1.5 x 9 cm) tubes or 10 ml to each of 10 sterile Pyrex milk dilution bottles and incubated in a 10% carbon dioxide-90% air mixture for 48 hours. Samples were taken from 2 or 3 cultures of each series for total counts which were determined by dilution and plating. These plates were incubated for 4 days in 10% carbon dioxide. Mutant counts were determined by direct plating of whole 1 ml cultures or aliquots of the larger 10 ml cultures and incubation of these plates in air. From the data obtained in these experiments mutation rates could be calculated from the total number of mutants or from the fraction of cultures containing no mutants using the formulas of Luria and Delbrúck as modified by Lederberg. The data given in this slide show very similar mutation rates for the nine strains tested. One of the strains, 6232C, was received by us with the history that it was CO2 stable; note that it has the lowest mutation rate of all the strains tested. The mean rate for this mutation is approximately 3 X 10- 1° per cell division. A much greater variation in mutant counts was found in a series of individual cultures than in replicate samples from the same culture, which indicate that the mutation is spontaneous and is not induced by the lower pCO2 of the air. Calculation of mutation rate from the total number of mutants depends on similar growth rates of mutant and parent in mixed culture where the ratio of mutant to parent is very small. To test growth rates under these conditions a flask of broth was inoculated with the parent Krause stock and a mutant strain derived from it. The initial ratio of mutant to parent was 1.0:107. After 48 hours incubation in 10% carbon dioxide the ratio was still 1.0:107 although total counts had increased 10-fold. Growth rates of the two strains in pure culture when incubated in 10% carbon dioxide were identical during the logarithmic phase of growth. It appeared possible that not all of the mutants were capable of forming colonies in the presence of large numbers of nonproliferating parent cells. To test this possibility a mutant strain from the parent Krause stock was plated in agar containing a background of 108 cells per ml of the Krause strain and in agar medium alone. The plates were then incubated in air. There was no significant difference in the counts of the two series, but the colonies of the mutant were much smaller when plated in the presence of large numbers of parent cells. In each of the mutation rate experiments, several colonies from the plates incubated in air were transferred to agar slants. These mutants which no longer required added CO2 for growth did not revert on serial subculture in either air or 10 per cent CO2. Cultures were tested for purity by Gram staining and high titer agglutination is anti-serum

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against Brucella abortus. Cultures were tested for colony type by the methods described by Henry and, more recently by Braun. Mr. White working in our laboratories has developed a method for detection of colonial variation that we find most useful. Cultures are streaked onto Albini brucella agar made to contain 1% glucose, 5% glycerol, and 2.5% agar. After incubation the plates are flooded with a dilute solution of crystal violet, the excess dye is removed, and the plates are observed with a low power dissecting microscope with the use of obliquely transmitted light. All of the cultures we used were smooth and the mutants derived from them were smooth. However, in order to show this technique for detection of colonial variants we took some kodachrome slides of other cultures of Br. abortus showing different colonial types. One of these slides presented the appearance of a normal smooth colony-the blue-green one-and a rough colony showing as red to purple. Another slide showed again a normal smooth colony as blue green and a mucoid colony which appears as dark purple with a cracked surface. Thus it is quite easy using this method that Mr. White has developed for a person with little experience to tell if he is working with a mixture of smooth and non-smooth organisms in a culture. In addition to the mutation studies, we have compared the metabolism of the parent and mutant for possible differences in metabolic pathways. The respiration rates of resting cell suspensions of the parent Krause strain and the mutant KC strain derived from it have been determined for a variety of substrates. There are no appreciable differences between parent and mutant strains in either rate of oxygen uptake determined as C0 2(N) or total oxygen consumed. These data indicate a fairly high degree of oxidative assimilation since the oxygen consumed per mole of substrate is considerably lower than that required for complete oxidation. The use of inhibitors revealed no differences in metabolic pathways in the parent and mutant strains. Sodium azide and 2,4-dinitrophenol in concentrations of M/600 and M/6000 respectively nearly doubled the rate of acetate oxidation and increased the total oxygen uptake to a value which approximated the amount required for complete oxidation. Acetate oxidation was inhibited by sodium arsenite and sodium bisulfide which indicates that alpha keto acids are intermediates in acetate oxidation in both parent and mutant. Conversely, the same concentrations of azide and dinitrophenol which stimulated the rate of acetate oxidation almost completely inhibited the oxidation of L-asparagine and L-glutamic acid. Asparagine oxidation was inhibited by an equimolar concentration of its enantiomorph while L-glutamic acid oxidation is not. No nutritional differences have been observed between parent and mutant strains other than the requirement for added carbon dioxide by the parent. The mutant strains examined thus far have been neither

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more nor less "exacting" than the parent strains from which they were derived. Earlier studies in our laboratories had shown that the CO2 requiring strains which we tested failed to grow in an Asparagine-Synthetic medium of Gerhardt and Wilson, while strains that did not require added C02 grew well in this medium. This indicated a correlation between the added C02 requirement and some other nutritional deficiency. We have found, however, that this nutritional deficiency is not linked with the CO2 requirement. Strains that require added C02 and that do not grow in the asparagine medium produce mutants that while they will grow in air on complex media fail to grow on the simplified asparagine medium. In so far as these experiments show mutant strains have the same nutritional pattern as their added C02 requiring parent. Also, we now have an added C02 requiring strain that will grow on the asparagine medium. Attempts to by-pass the added C02 requirement by the addition of some of the known C02 fixation products have not been successful. We have tried some of the Krebs intermediates and related amino acids, as well as adenine, quanine, and uracil. Culture filtrates and cell autolysates also failed to replace the added C02 requirements. CONCLUSIONS The results indicate that Brucella abortus loses its requirement for added carbon dioxide by spontaneous mutation at a rate of approximately 3 X 10 - 1° per cell division. Training of such cultures to dispense with their carbon dioxide requirements probably consists of a selection of spontaneously occurring mutants rather than gradual adaptation of the whole culture. The low mutation rate is indicative of a high degree of stability for carbon dioxide requirement in cultures of Br. abortus. The data on oxidative metabolism of various substrates associated with the Krebs cycle indicate a basic similarity of dissimilatory reactions in both parent and mutant types. This is confirmed by the similar behavior in the presence of various metabolic inhibitors. Thus far we have no information concerning the biochemical basis of the added carbon dioxide requirement of Br. abortus. Gerhardt and Wilson have demonstrated that some Krebs cycle intermediates do not by-pass the added carbon dioxide requirement. This does not necessarily preclude a basic biochemical similarity between the added carbon dioxide requirement and the "normal" heterotrophic requirement since several workers have reported that Krebs intermediates do not completely replace carbon dioxide. Currently we are using radioactive carbon in C02 to study this problem and we are comparing C02 fixation by the parent and by the mutant. We hope to report data from these experiments in the near future.

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DIOXIDO DE CARBONO QUE NECESITA LA BRUCELLA ABORTUS (Sumario) Se encontró que grupos pequeños de células en cultivos de cepas de Brucella abortus que requieren la adición de CO2, podían proliferar sin dicha adición. El análisis de la distribución de estas células es una variación mucho mayor en cultivos individuales que en muestra de control del mismo cultivo. Esto sugiere enfáticamente una mutación espontánea debida a que desaparece la necesidad de agregar C02. La proporción de esta mutación-de la necesidad de adición de CO 2 a la habilidad de poder crecer en el aire-ha sido calculada de la fracción de cultivos que no contenían mutantes (en una serie de cultivos similares) y del recuento total de mutantes de toda la serie. Para las cepas probadas, la proporción es aproximadamente de 5 X 10-1 ° por división de célula. Se realizó una comparación del metabolismo de célula en descanso de cepas mutantes y originarias, usando el microrespirómetro convencional de Warburg. En la proporción de oxidación por mutante y originaria de una variedad de substratos, no se ha observado diferencia alguna. El uso de inhibidores ha indicado modelos metabólicos muy similares. Las células en descanso, ya sean mutante u original, asimilan acetato y maletato por oxidación. La asimilación de acetato por ambas cepas es inhibida casi completamente por "azide" de sodio o dinitrofenol al 2.4. En cuanto a las necesidades de nutrición no se han observado otras diferencias aparte de la necesidad de agregar CO2. Se ha tratado de eliminar la necesidad de agregar CO 2, pero no se ha tenido éxito en este sentido.

USE OF THE EMBRYONATING EGG FOR ISOLATION OF BRUCELLA IN A PUBLIC HEALTH DIAGNOSTIC LABORATORY: A YOLK SAC TECHNIQUE By

KATHLEEN GAY,

B.S.,

AND S. R. DAMON, PH.D.

Senior Bacteriologist, and Director, Bureau of Laboratories, Indiana State Board of Health, Indianapolis, Indiana INTRODUCTION

Examinations for Brucella in the public health laboratory are generally made by culture or animal inoculation of blood specimens. The limitations of these technics are obvious to all who have had occasion to apply them and it is evident that a procedure which will permit more rapid reporting of results to the physician and also yield a higher percentage of recoveries is badly needed. Goodpasture and Anderson,' Buddingh and Womack,2 Ruiz-Castaneda, 3 and others have shown the causative organism of brucellosis to be preferentially an intracellular parasite. It seemed to the authors, therefore, that the embryonating egg might be useful as a medium for primary isolation. A survey of the literature revealed only a few specific references to the cultivation of Brucella in developing chick embryos; and of these reports, the majority dealt with the use of the fertilized egg as a means of evaluating therapeutic agents or as a technic for studying the pathology and invasive ability of Brucella. No reports of its use as a laboratory diagnostic aid were found. In undertaking a study of the value of the embryonating egg, the yolk sac inoculation technic was selected as the logical one to employ, since this area can receive a sizeable amount of blood without significant harm to the embryo, and the yolk itself is highly nutritious. The first step was to determine that age embryo for receipt of the inoculum which would provide optimum conditions for multiplication of small numbers of organisms. A preliminary experiment 4 indicated that 1-2 day embryonating eggs were of very doubtful value and that the three to six-day embryos provided optimum conditions. Three, four, and five-day embryos were finally selected for an evaluation series because they not only worked into a practical scheme for inoculation but also provided 9-11 days of further embryo development for multiplication of Brucella, before the yolk was so reduced that it was difficult to manipulate. The purpose of this paper is to describe a method which is proving successful, and to present experimental evidence for the selection of certain technics to be used in an evaluation of the three methods of isolating 157

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Brucella from the blood stream, namely, guinea pig inoculation, culture, and growth in the egg embryo. MATERIALS AND METHODS

Specimens.-Clots from blood specimens received routinely at the Indiana State Board of Health for diagnostic agglutination tests were employed. The sera were first examined for Brucella agglutinins with a commercial Brucella abortus rapid slide test antigen. If there was complete agglutination at the 1:20 dilution level, the clot was divided for culturing, guinea pig inoculation and egg injection. Part of the clot was injected directly into a tube of modified Huddleson crystal violet tryptose broth. The portion to be received by eggs and guinea pigs was forced through a syringe into bottles containing 5-7 ml tryptose broth and several small glass beads. These bottles were stoppered and shaken three minutes for further disintegration of the clot. Eggs.-White leghorn "hatching quality" eggs (avg. wt. 59 gm.) were obtained weekly from a local poultry supply house and held at 20°C until the day of incubation. That is, a week's supply was received on Wednesday and a sufficient number incubated each Monday and Friday, so that each day of the week 3-, 4-, or 5-day old live embryos were available. Before and after injection, all eggs were kept at 98-99°F (37°C-±), relative humidity 50-560, in an electric hatchery incubator. They were not turned at any time since doing so was found nonessential to the technic and also complicated the procedure for locating the yolk sacs. Candling of Eggs.-A small egg candler with a 100 watt bulb was installed in a plywood box 20" square. The interior was painted black and one side left entirely open to receive the egg racks. The opening was covered with a photographer's heavy black cloth. With this device it was possible to accurately candle embryos as young as three days. All eggs were candled immediately before use and the position of the embryo marked. Inoculation of Eggs.-Eggs which had incubated 3-5 days were first candled and those containing live embryos selected for inoculation. A small hole was drilled into the large end of each egg with an electric table model dental drill and § 22 S.S. White abrasive point. With a 20-gauge 12" hypodermic needle 0.5 ml of the blood-broth specimen was injected into each yolk sac. In making the inoculation the egg, with the marked embryo at the top, was placed horizontally on the table and the needle was inserted toward the center for 0.5-0.75". If the embryo was 3 days old, the direction of the needle was slightly upward; if 4 or 5 days, directly inward toward the center. Inoculations were made the day of the agglutination tests, although an occasional clot was held over the weekend. When this was done, the clot structure was not disturbed until the day of the inoculation.

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The area around each hole was swabbed before and after inoculation with tincture of iodine and the hole finally sealed with a swab of collodion. Examination of Eggs.-The examinations consisted of three subcultures from the yolk at about five day intervals and a final harvesting of all live embryos on the 18th or 19th day of total incubation, the embryo ages at each subculture being 9-10, 14 and 18-19 days, respectively. Several drops of yolk material from each egg were withdrawn and placed on dry tryptose agar (pH 6.6-6.7) plates, and streaked over the surface with a wire loop or sterile swab. All plates were held two weeks in an incubator with a C02 concentration of approximately ten per cent maintained at all time. If any embryos remained viable on the 18th or l9th day of incubation they were harvested and portions of the blood, yolk sac wall, internal organs, and mixed fluids streaked onto tryptose agar plates. Withdrawal of Yolk.-The yolk was withdrawn in a manner similar to that of the injections. However, since the position of the yolk sac gradually changes with further development of the embryo, allowances must be made for this by inserting the hypodermic needle at different angles and depths. When the egg is placed horizontally on the table, as a rule, the older the embryo the more the yolk position shifts from a high anterior position to a low posterior one. For the first withdrawal, when the embryo was 9-10 days old, the needle was inserted as for inoculation of a five-day embryo. However, for the second withdrawal at 14 days, the needle was inserted deeper and angled downwvard about 30 degrees. Occasionally, further probing was necessary to find a misplaced yolk. Identification of Brucella.-Suspicious colonies were fished to tryptose agar slants and incubated in the CO 2 incubator for 24-48 hours. A spot agglutination test was then made from the growth on the slant, using a commercial Br. abortus antiserum. If clumping occurred, duplicate tryptose agar slants were inoculated, one incubated in air and one in CO2 to check CO2 growth requirements. A saturated lead acetate paper was placed in the mouth of the latter to determine H 2S production. Growth from the original slant was also streaked onto basic fuchsin and thionin dye plates (dye conc. 1:100,000) for species typing. The remaining growth was then suspended in 0.5% formalinized saline, diluted to the proper density, and tube agglutination tests set up, with both the commercial antiserum and the homologous serum from the patient. EXPERIMENTAL RESULTS The first specimens of blood injected into eggs came from autopsied guinea pigs which had been inoculated subcutaneously with human blood from suspected cases of brucellosis, and from swine experimentally infected with Brucella. Fifteen specimens were injected into 3- and 5-day

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old eggs and three yolk subcultures made as previously described. Penicillin G was used in half the eggs in an attempt to control contamination. Four recoveries were obtained, all Br. suis strains-one from the guinea pig and three from the swine bloods, though only from eggs not receiving the antibiotic. In addition, a Br. melitensis strain was recovered from the blood of a treated ambulatory case when the clot was inoculated into 3-day old eggs. At this point it seemed desirous to have selected specimens in a number sufficient to make an evaluation test of the chick embryo method. Therefore, it was decided to select blood clots from those specimens being received routinely in the laboratory for agglutination tests. Experiment 1.-The specimens were treated as described in the foregoing section, except that any clots from sera showing even a partial agglutination were inoculated, three eggs receiving the blood clot in a low concentration of penicillin broth and three receiving the blood without the antibiotic. Seventeen specimens were injected. There were three yolk subcultures. Embryo tissues were not examined, otherwise. Thirteen specimens were negative, two unsatisfactory due to heavy contamination, and two yielded isolations of Br. abortus. Multiplication of Brucella was not detected in those eggs receiving penicillin except in the instance of a single embryo in which growth was quite delayed. By this time it was apparent that the low concentration of penicillin used did not control contamination and it also appeared that the antibiotic was inhibitory for Brucella. Consequently, any further attempt to control contamination was abandoned. Experiment 2.-In this group no penicillin was used. Each blood clot was injected into six eggs. As in Experiment 1 three yolk subcultures were made and the embryos were not harvested for a final examination. Sixteen clots were injected, with no recoveries and one unsatisfactory contaminated specimen. At this point, it was decided to employ more highly selected specimens, reduce the number of eggs per specimen and begin an evaluation series comprising inoculations into a broth for culture, into the guinea pig and into the eggs, using altogether the materials and methods described in the preceding section. The number of eggs per specimen was reduced from six to four since observations already made indicated this was probably a safe minimum. Further, as an additional examination of the eggs before discarding them, embryo tissues as well as the yolk were to be cultured. Experiment 3.-Each clot of this series was examined by inoculation subcutaneously into guinea pigs, inoculation into a tube of modified Huddleson's crystal violet tryptose broth (final dye concentration 1:1,000,000) from which periodic subcultures were made, and into embryonating eggs 3-5 days of age. Seventy-five specimens have been inoculated and examined as described in Materials and Methods. Seventeen were discarded as unsatisfactory due to con-

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tamination of the blood clot. From the 58 unsatisfactory specimens, there were 5 isolations, all Br. abortus. Three of the five recoveries were not obtained by either of the other methods. All five isolations were obtained by the seventh day following inoculation of the eggs, although an occasional egg of a group from which an isolation was made on the first yolk subculture did not show growth until the second withdrawal. Colonies of Brucella came up on the second day of the agar plate incubation. All five strains were typically Br. abortus. Table 1 summarizes all the recoveries made from blood clots inoculated into the yolk sacs of 3-5 day embryonating eggs. It is to be noted that TABLE 1.-The recovery of Brucella from blood clots Age of Embryo (days) Type

Lab. No.

Specimen Source

Titer*

At Injection

At Isolation

swine

5

13

12 19 5973

swine swine guinea pig

5 5 3

13 10 8

Melitensis

6356

human

1:320

3

12

Abortus

3338 3953 4046 4138 2172

human human human human human

1:320 1:160 1:80 1:320 1:160

4 4 3 4 4

9 10 10 9 9

Suis

2

* The highest serum dilution in which a 2+ agglutination reading was obtained.

only 5-9 days of incubation were required for isolating these Brucella strains. DISCUSSION

Although previous experiments have shown Brucella to multiply rapidly in 3-10 day old embryos, the optimum age range has been shown to be 3-6. The younger ages, 3-5 days, were selected for injection because in addition to providing optimum conditions for the growth of Brucella, they permit inoculation at that time when the yolk begins to swell. It is too early in the course of the experimental series to state that any one age embryo at the time of injection is superior to another; as recoveries have been made from all three ages employed. Attention has already been called to the fact that the final harvesting of the embryos and the third subculture of yolk material has not yielded additional isolations. Although these data have not been included in the experi-

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mental results, with the exception of half a dozen eggs, all live embryos at the pre-hatching stage were examined by staining tissue smears with Macchiavello's rickettsial stain.5 No additional infections were found. The fact should be mentioned that not all the specimens injected came from frank clinical cases. Although they usually came from a patient showing a fairly high titer of Brucella agglutinins, a significant percentage came from treatment checks and so-called chronic cases. Based on observations and results of the experimental data presented in this paper, the following procedure, which is applicable to use in any public health diagnostic laboratory and is now proving of considerable value in the hands of the authors, is suggested as one yielding a more rapid and efficient recovery of Brucella from the blood stream. It utilizes a simple type of specimen, which any physician prefers submitting. It provides for a preferentially-intracellular parasite the living tissue cells as in the guinea pig, without the concomitant danger to laboratory personnel and the expense and upkeep of animals; while at the same time it is a unit of culture medium which can be nearly as easily handled as the tube of broth. It can reduce the time interval between taking the specimen and receipt of the result by the physician to ten days in contrast to three or four weeks. The following scheme is suggested for laboratory use as a routine diagnostic aid: Eggs may be purchased once a week from any supply house handling "hatching" quality eggs and, if necessary, kept at least a week at approximately 20°C before incubating. If the eggs are set twice a week, that is, on Monday and Friday, either a 3-, 4-, or 5-day old embryo will be available each day except Sunday for inoculations. If the specimen is in the form of a clot, break up as previously described before injecting. Four eggs, as determined by the authors, is probably the minimum number per specimen to use. On the fourth or fifth day after inoculation withdraw a small portion of the yolk and streak onto a non-inhibitory plating medium, incubating the plates in an increased C02 atmosphere. On the fourteenth day, make a second and final yolk subculture. CONCLUSIONS 1. A method for using the embryonating chick egg for primary isolation of Brucella from the blood stream has been presented and supporting experimental data given. 2. Essentially, this technic consists of injecting specimens directly into the yolk sacs of 3-, 4-, and 5-day old embryos and subculturing periodically. 3. All three types of Brucella have been isolated and in many instances this technic has proved superior to guinea pig inoculation or culture.

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4. The methods described are being used in an evaluation of guinea pigs, eggs, and culture media for the isolation of Brucella from clotted blood. 5. The advantages of the egg embryo yolk sac technic are: (a) it provides living tissue cells in a compact medium; (b) it permits recovery of all three types of Brucella; (c) it gives quick results; (d) it is relatively inexpensive; and (e) it entails a minimum amount of danger for laboratory personnel. REFERENCES (1) Goodpasture, E. W., and Anderson, K.: 1937. The problem of infection as presented by bacterial invasion of the chorio-allantoic membrane in chick embryos. Am. Jour. Path., 13S 149. (2) Buddingh, G. J., and Womack, F. C.: 1941. Observations on the Infection of Chick Embryos with Bacterium tularense, Brucella, and Pasteurella Pestis. Jour. Exp. Med., 74: 213. (3) Ruiz-Castañeda, M.: 1947. Studies on the pathogenesis of brucellosis. Proc. Soc. Exptl. Biol. and Med.,64: 298. (4) Gay, Kathleen, and Damon, S. R.: Use of the chick embryo for isolation of Brucella: Multiplication of the organism in the yolk sac and selection of the embryo age optimal for isolation from blood. In press. (5) Smith, D. T., and Martin, S. M.: Zinsser's Textbook of Bacteriology, 9th ed., 1948. Appleton-Century-Crofts, Inc. AISLAMIENTO DE LA BRUCELLA MEDIANTE EL USO DEL HUEVO EN ESTADO DE EMBRIóN EN UN LABORATORIO DE DIAGNóSTICO (Sumario) Aunque desde hace algún tiempo algunos laboratorios de salud pública han estado interesados en el problema del aislamiento de la Brucella de muestras de sangre, muchos de ellos no han introducido todavía en sus prácticas de diagnóstico los procedimientos de cultivo de inoculación de cobayos. La vacilación en cuanto a la introducción de estos procedimientos se debe posiblemente al bajo porcentaje de los aislamientos de los tipos de especímenes generalmente sometidos, así como al riesgo que representan para el personal cuando se utilizan animales en el laboratorio. Durante algunos años, en el laboratorio de diagnóstico de la Junta de Salubridad del Estado de Indiana, se han empleado métodos de cultivo para el aislamiento de la Brucella. Sin embargo, desde la iniciación del Proyecto de Estudio de la Brucelosis en el Estado de Indiana, un aspecto que pareció merecer investigación fué si habría o no otros medios de aislamiento de estos organismos, medios que habrían de ser adaptables a la rutina de diagnóstico de laboratorio, y al mismo tiempo de valor igual o mayor que los métodos empleados en la actualidad. Por lo tanto, se consideró la posibilidad de usar huevos de gallina en embrión, como medio regular de cultivo. Los trabajos de Goodpasture y sus colaboradores han demostrado desde hace mucho tiempo que la Brucella puede sobrevivir y multiplicarse en huevos, en tanto que Spink y otros han utilizado embriones de huevos inoculados para probar diversos agentes terapéuticos, a pesar de no

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existir en forma impresa ninguna información relativa al uso del embrión de huevo como medio de cultivo para diagnósticos. A través de investigaciones preliminares, pronto se demostró el valor de los huevos en embrión como medio de cultivo; en consecuencia se seleccionaron embriones incipientes-de 3, 4 y 5 dias-que presentaban condiciones óptimas para la multiplicación de los tres tipos de Brucella. Además, pareció más ventajoso la inoculación de la muestra de sangre a través de la membrana vitelina. En sintesis, el procedimiento ha consistido en utilizar jeringuillas de 5 ml sin agujas, para introducir los coágulos de sangre en frascos con 7 ml de caldo de triptosa y bolitas de vidrio esterilizadas. La agitación de los frascos por 2 6 3 minutos sirve para desintegrar más los coágulos, después de lo cual se inyecta 0.5 ml en la membrana vitelina de cada embrión de huevo; se utilizan cuatro de éstos. La incubación de los huevos inoculados se hizo a 37°C, y después de 4

y 8 días se extrajeron porciones de yema para su cultivo en láminas con agar triptosa simple. El subcultivo final de la yema de los huevos se hizo después de 15 días de incubación, en esta oportunidad se recogieron los embriones con vida, y también se extrajeron y cultivaron el hígado, el bazo, la membrana vitelina, la sangre, el corazón y la vesícula biliar. Las láminas inoculadas se incubaron a 36°C, en una atmósfera de CO 2, y los organismos que surgieron fueron identificados mediante cultivo y aglutinación. Se han aislado las tres variedades de Bruecella de los coágulos de sangre mediante el método de inoculación de huevos de gallina en embrión. La técnica seguida es relativamente sencilla, y pueden obtenerse resultados con mayor rapidez que mediante cultivos o inoculaciones en cobayos.

DIAGNÓSTICO BACTERIOLÓGICO Y BIOLÓGICO DE LA BRUCELOSIS Por Luis A. PHILIPPS Jefe, Instituto Nacional de Biologia Animal, Ministerio de Agricultura, Lima, Perú El diagnóstico bacteriológico y biológico de la brucelosis es un problema de vital importancia; tanto en la medicina humana como en la medicina veterinaria. En el campo de la salud pública el problema reviste caracteres de vital importancia, si se tiene en cuenta que el hombre puede ser infectado por cualquiera de los tres tipos de Brucella conocidos y un diagnóstico rápido puede asegurar el tratamiento adecuado. En el campo veterinario, cuando se trata de aplicar un programa de erradicación de la brucelosis con miras a evitar la difusión de la brucelosis humana y aumentar la producción animal evitando los casos de aborto y de esterilidad. Realizada la siembra de un material sospechoso siguiendo las técnicas ya establecidas y sospechosa una colonia de Brucella es repicada en dos medios denominados "IM" y "SMG". Estos dos medios que fueron ideados por el bacteriólogo peruano, Dr. Hector Colichón, para el despistaje de colonias de tipo intestinal deben su nombre, el de IM a las dos primeras letras del Instituto "IMEX" (Instituto de Microbiología Experimental) del cual es Director el citado Doctor; el segundo, a su composición primordial: subacetato glucosa, manita (SMG). En nuestra práctica de laboratorio nosotros los hemos utilizado en una forma más amplia sirviéndonos para el despistaje de colonias pertenecientes a la familia Parvobacteriaceae; siendo las reacciones bioquímicas características que se traducen en cambio de color de los medios por la alcalinización o acidificación de ellos o por la fragmentación del medio sólido (SMG) y recolección de gas en la campanita del medio de "IM", cuando hay ataque de los azúcares hasta la descomposición de ellos con la consiguiente producción de gas. La composición de estos dos medios es la siguiente: "IM" Agua de peptona (ph 7.4) .................................... 100 cc

Mezcla triple carbohidrato (1)...............................

8 cc

1 cc Indicador de "IM" (2) ....................................... 5 cc Urea al 20% ................................................ (1) Mezcla triple carbohidrato: Lactosa al 20% .......................................... 75 cc 3.5 ce Sacarosa al 20% ......................................... 3.5 ce Manita al 20% .......................................... (2) Indicador de "IM" Es una mezcla de indicador de Andrade y bacto timol azul. Afiadiendo a 100 cce de este indicador 0.4 gm de bacto timol azul. 165

166

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

El agua de peptona se prepara con proteosa-peptona No. 2 Difco; cualquier otra peptona puede no ser adecuada; el uso de bacto peptona Difco no es recomendable para esta preparación. Disolver 10 gm de proteosa-peptona en 1000 cc de agua destilada (preferir bi-destilada) y luego agregar 5 gm de cloruro de sodio (NaC1) químicamente puro. Neutralizar y precipitar en caliente, filtrar por papel y ajustar a pH 7.4; repartir y luego esterilizar a 1 atmósfera por 15 minutos. Se emplea carbohidrato marca Difco o Merck y la esterilización se hace por filtración. El indicador de "IM" sirve para demostrar las variaciones alcalinas como las ácidas en los cultivos. El indicador de Andrade se prepara con 0.2 gm de bacto fuchsina ácida (National Aniline Div. Dye content 5.8%) disuelto en 92 cc de agua destilada, a la que luego disuelta se agrega 8 cc de NaOH normal para decolorarla. La urea igualmente debe ser esterilizada por filtración y se debe emplear urea químicamente pura. Preparación para el uso.-De acuerdo con las cantidades especificadas se agrega al agua de peptona la correspondiente cantidad de carbohidrato, luego el indicador y por último la urea. Agitar y repartir en tubos esterilizados como los usados para observar la producción de gas (Durham). El llenado de las campanitas se hace mediante una bomba de vacío, teniendo la precaución de calentarlos antes de someterlos a la cámara de vacío a fin de obtener en los tubitos interiores el lleno completo (todo indicio de burbuja obligará a desechar el tubo o a repetir el vacío en caliente). MEDIO "SMG" (Manita, glucosa-subacetato de plomo) Bacto-Tryptona Difco ................................ 15 gm Bacto-Proteosa Peptona Difco ........................ 5 gm 0.5 gm Subacetato de plomo (reactivo de Merck) ............. Tiosufalto de sodio (reactivo de Merck) ............... 0.2 gm Bacto-Manitol Difco .................................. 10 gm (50 cc sol. al 20%) Bacto-Dextrosa Difco .................................. 1 gm (5 cc sol. al 20%) Solución de rojo de fenol al 0.2% ..................... 15 ce Bacto-Agar Difco ................... 10 0................. gm Agua destilada ....................................... 1000 cc

Preparación.-Disolver las sales en el agua destilada, en seguida agregar las peptonas. Ajustar el pH a 7.1 y luego agregar la solución de rojo de fenol. Este medio no se filtra. La mezcla se esteriliza a 1 atmósfera durante 15 minutos. Al sacarlo del autoclave aun caliente se agrega asépticamente los carbohidratos, usando soluciones al 20% esterilizadas por filtración. Repartir con esterilidad en balones de 100 a 200 cc para uso cotidiano.

DIAGNÓSTICO DE LA BRUCELOSIS

167

La solución de rojo de fenol se prepara disolviendo un gramo de rojo de fenol en 40 cc de NaOH N/10 completando luego a 500 cc con agua destilada. Preparación para el uso.--Fundir el medio en baño de María a ebullición, dejarlo enfriar a 600C y luego repartir en tubos de 10 x 100 mm estérilmente y dejarlo endurecer en posición semi-inclinada; verificar un control de esterilidad antes de su uso. En estos dos medios la reacción de la Brucella es inconfundible. El medio de "IM" originalmente es de color amarillo, la producción de acidez se traduce en viraje de color desde el rosado al rojo y la alcalinidad por el viraje al azul. El medio "SMG" es de color rosado y la producción de acidez se traduce en variación al amarillo y la alcalinidad hacia el rojo. La producción de H 2S se revela por el ennegrecimiento del medio. CUADRO 1.-Distintas reacciones en los medios de "IM" y "SMG" de gérmenes cuyas colonias son similares a las de Brucellas 1"IMI"

"SMG"

Tipo bioqufmico

Pasteurella .........

Alc.

Ac.

-

Lig. Ac .

Streptococcus ....... Hemophilus ......... Alc. Brucella ............. Alc. Alcaligenes ..........

Ac. -

-

Gas

A

Alc.

Ac.

-

_ _ _Alc. Alc.

Gas

H2S

Lig. Ac.

-

-

Ac. Lig. Ac. -

-_ -

-

_ -

Alc. = Alcalino; Ac. = Acido; Lig. Ac. = Ligeramente ácido. DIFERENCIACIóN DE LAS BRUCELLAS ABORTUS, MELITENSIS Y SUIS POR EL USO DE DISTINTOS COLORANTES

Por la observación del siguiente cuadro que corresponde a un estudio comparativo de distintas cepas en lo referente al límite de tolerancia para su desarrollo en distintas concentraciones de colorantes, se puede concluir que en el tipado s61o son necesarios las siguientes concentraciones de colorantes: tionina al 1/30,000, fuchsina al 1/25,000 y cristal violeta al 1/50,000. Las demás concentraciones de tionina, fuchsina y cristal violeta no son necesarias. En la pironina las distintas cepas desarrollan en forma similar. En el cuadro siguiente las cepas numeradas del 1 al 9 corresponden a Br. suis; del 10 al 15 a Br. abortus y del 16 al 18 a Br. melitensis. DIAGNóSTICO BIOLÓGICO

Entre los distintos procedimientos de diagnóstico biológico la intradermo reacción es deficiente, ya que he podido observar que animales con reacción francamente positiva a la aglutinación tenían una intradermorreacción negativa; igualmente animales con aglutinación nega-

168

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS CUADRO 2 Tionina

Cepa

Fuchsina

No. 1/30,000

1 2

-++ ++ ++ ++ -++ -++ ++ ++ +-q +-q +-q

3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18

1/50,000

++ ++ ++ ++ ++ ++ ++ ++ ++

1/25,000

1/75,000

Cristal violeta

1/50,000

1/100,000

1/50,000

I

++ ++ ++ ++ ++ ++ ++ ++ q-q ++ q-q +-q

I I

1

++ ++ ++ q-q + +-q +

+

++ ++ +-q +-q +++ +q-

+

+

+

+++++++q+-

+

CUADRO 3 Piromina

H.S

Cepa No. 1/100,000

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15

16 17 18

++ ++ q-q ++ q-q ++ q-q ++ q-q ++ q-q ++ q-q ++ ++ + +f + + + + + + +

1/200,000

++ ++ ++ ++ ++ ++ ++ q-q ++ q-q +++ +-q ++ q-q + q-q +

+ q++q+ q+ ++ +++++-

+

tiva mostraban la intradermo reacción, unos positiva y otros negativa. Estas aseveraciones se ponen de manifiesto en el cuadro siguiente. La opsonocitofagia no es aconsejable para el diagnóstico de la bruce-

169

DIAGNÓSTICO DE LA BRUCELOSIS CUADRO 4.-Aglutinación introdermorreaccióny opsonocitofagia No. del Animal

ATlítuln o die Aglutinaci6n

Marcada

1921 2775 0034 2668 2568 2519 1926 2312 2423 1998 1450 1449 4320 6282 2156 2549 2261 2211 2580 1781 2437 2619

1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25

0501 1608 2356 0171 2302 2240 2511 1620 2498 8049 2771 2508 6304 1562 2212 2774 5658

1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50

o 1 o 1 112

5506 1627 0112 6379 1966 1495 2358

1/100 1/100 1/100 1/100 1/100 1/100 1/100

O 5 13 2 o 10 o

Moderada

Pequeña

Mo ,d

12 5 5

3 2 3 1 10

7

1 o 2 o 9 6 2 o 8 3 3 o 1 6

8 12 11 15 6 11 10 3 3 8 4 2 2

12 7 12 8 O 9 9 5 6 15 10 20 16

+

+ +++ ++ + ++ +++

7

9

+

3 9 9

20 9 12

+ +

8 1 2 3 15

16 14 3 16 2 8 1 21

++ ++ ++ ++ ++ ++ +

1 7

4 6 2 4 1 7

7

o 8 o 5 5 o 4

2

4

3

j

4

1 4 4

7

6

Intradermorreacción

++

3 10 5 6 3 o

o

No. índice Opsónico

11 9 8 3 6 17 3 14 25 11 24 6 13 13 22 6 19 14 24 17 6

7

4

Negativa

1

+ + + ++ ++ ++ + ++ + ++ + +

+

+++ +

+++ ++

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

170

CUADRO 4.-Agglutinación introdermorreaccióny opsonocitofagia (Cont.) No. del

Animal

Título de

Aglutinacideón

Marcada

Moderada

Pequefia

Negativa

No. índice

Intradermo-

Opsónico

Intradermoeacción

2481 2129

1/100 1/100

0 0

1 1

6 3

18 21

19 10

+ +

8052

1/400

4

8

3

10

43

+++

2532

0/0

4

7

10

4

47

-

CUADRO 5 Títulos de Aglutinación Antígeno*

Suero 1/25

2870

1/50

1/100

1/200

1/400

+ 1

2516

+

+ 2965

2540

+

+ + + + + + + + + + + + + + + +

I I I I I

+ + + +

q++ +++ + +-

171

DIAGNOSTICO DE LA BRUCELOSIS CUADRO 5.-(Cont.) Títulos de Aglutinación Antigeno*

Suero 1/25

2999

2351

1/50

1/0l

1/200

1/400

+

1 1 1

1 1 1 1 3195

3200

3198

* Las fechas de preparación de los antigenos fueron: A-enero 2 de 1941; B-junio 26 de 1942; C-julio 30 de 1943; D-abril 1 de 1944; E-abril 26 de 1945; F-marzo 12 de 1946; G-abril 16 de 1947; H-noviembre 15 de 1948. En las lecturas de las aglutinaciones de estos distintos sueros se observa que los antígenos no han sufrido cambio alguno en sus propiedades de aglutinabilidad.

172

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

losis ya que no hay ninguna relación entre el índice opsónico y el título de aglutinación, pues en el cuadro que antecede se puede observar que animales que tienen un título de aglutinación elevado poseen un índice opsónico bajo y a la inversa. Las aglutininas y las opsoninas son dos entidades completamente diferentes. En la opsonocitofagia se han seguido las técnicas de Keller, Pharris, Crit y Gaub consignadas en el libro de Métodos de Laboratorio Clínico de Kolmer y Boerner 1948. El CUADRO 6.-Estudio comparativo de la pruebas del anillo y las aglutinaciones del suero hemático y suero lácteo

·

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Pruebas del anillo (ring test)

++++ ++++ +++ ++ +++ ++++ +++ ++++

++ ++++ ++ ++++ ++ +++ ++ ++ +++ ++

I

Aglutinaciones No. S. Hemático

Suero Lácteo

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

1/800 1/50 1/100

1/200 1/25

1/50 1/100 1/50 1/100 1/200

1/200 1/200 1/50 1/50 1/50 1/400 1/50 1/100

42

43 44 45 46

1/100 +

1/50

1/200