Bull. Org. mond. Sante 1959, 20, 1033-1051 Bull. Wld Hlth Org. IMMUNIZATION AGAINST BRUCELLA INFECTION 7. Immunological and Epidemiological Studies in Cordoba, Spain* SANFORD S. ELBERG, Ph.D. Professor ofBacteriology, University of Calijornia, Berkeley, Calif., USA WHO Consultant SYNOPSIS The author describes a study, carried out in the Province of C6rdoba, Spain, to test the efficacy of a live vaccine prepared from the Rev I strain of Brucella melitensis against caprine brucellosis and to determine the extent of natural infection in goats and humans in the Province. It was found that the vaccine significantly increased the resist- ance of the goats to infection without inducing a carrier state of the vaccine strain and that the immunity persisted for at least 15 months-the period of test. Serum agglutination tests, milk ring tests, and milk culture tests on goats showed that approximately 16-29% of the individual animals examined would be considered infective on the basis of one or other of the tests. Of the 1 8 herds tested, I I I were discovered to be harbouring infected animals. Serum agglutination tests on humans revealed that 25 of the 880 people tested (2.8%) had titres of 160 International Units (IU) or above and that, on the basis of a diagnostic titre of 80 IU or above, 7% of the population would be regarded as showing evidence of a past or present infection. Under an agreement between the Government of Spain and the World Health Organization for a study of the brucellosis problem, an invitation was accepted to carry out a small-scale study of the immunizing effective- ness of the Rev I strain of Brucella melitensis in Spain at the laboratories of the Instituto Provincial de Sanidad, Cordoba. The purpose of this study was to determine the 50% infective dose (IDr0) of a virulent challenge strain of Br. melitensis in immunized and non-immunized goats in the Cordoba area. Earlier studies on adult goats obtained from Texas demonstrated that a single subcutaneous injection of the live vaccine strain Br. melitensis, Rev I, prevented abortion and infection by a dose of the virulent challenge * These studies were carried out in 1957-58 during the author's tenure of a John Simon Guggenheim Fellowship and were supported in part by funds made available through an agreement between the Govern- ment of Spain and the World Health Organization and in part by funds from grant E-22, National Institutes of Health, USA. 788 -1033- 1034 S. S. ELBERG strain which induced abortion in 9 out of 12 and infection in 10 out of 12 non-immunized animals (Elberg & Faunce, 1957; Elberg & Meyer, 1958). Confirmatory experiments were reported by Jones, Thomson & Alton (1958), who found that the ID50 of goats immunized with Rev I strain was 62 times greater than that of non-immunized animals on the basis of one or more colonies recovered from any tissue and 750 times greater on the basis of generalized infection, i.e., one or more colonies recovered from any tissue other than the atlantal, parotid, retropharyngeal and submaxillary lymph-nodes-the so-called " regional " nodes draining the site of inocula- tion. Unpublished data of Alton (1958) from an experiment with the Rev I strain carried out in Malta further demonstrated the ability of the strain to protect a highly significant number of goats against infection and abor- tion under semi-field conditions. Materials and Methods Vaccine The Rev I strain of Br. melitensis was employed in these studies. This strain had been isolated as a streptomycin-independent mutant from a population of streptomycin-dependent Br. melitensis cells and has been fully described (Herzberg & Elberg, 1955; Elberg & Faunce, 1957). The strain was cultivated on Albimi agar, and harvested in Zobell solution (Zobell & Zobell, 1932). A viable plate count on Albimi agar was performed on this suspension. While the plates were incubated at 37°C for the 4-5 days necessary for growth of colonies of the vaccine strain, the suspension was stored at 5°C. When the number of viable cells per ml of suspension had been determined, the suspension was diluted in sterile Zobell solution to contain 1 x 109 cells per ml. Each goat received 1 ml of the diluted suspension subcutaneously in the suprascapular area. Challenge strain A suspension of Br. melitensis, strain 6015, was employed to establish brucella infection in the animals. This strain has been in constant use for this aspect of the work and its ID50 dose for Texas goats was 25 000 cells when last determined. Inasmuch as its virulence for mice and guinea-pigs has remained unchanged over the past several years it was assumed that the virulence for the goat would also be unchanged. Agglutination tests The standard antigen of the Agricultural Research Service, United States Department of Agriculture, was employed for all tests on the labo- ratory experimental animals. It was used in a dilution which yielded a titre of 1: 1000 with a sample of the International Standard Anti-Brucella abortus Serum made available for this work by the Central Veterinary Laboratory, Weybridge, England. IMMUNIZATION AGAINST BRUCELLA INFECTION 1035 For agglutination tests on animal sera gathered from the various herds in the Province of Cordoba, a portion ofBr. melitensis antigen, commercially available, was employed after standardizing it against the sample of the International Standard. Thus all agglutination titres are expressed in terms of the International Unit (IU). Complement-fixation test The modification of the Kolmer test designed by Pirosky (1941) was used in the latter part of this study. The antigen was prepared by Dr Julio G. Barrera Oro, Instituto Nacional de Microbiologia, Buenos Aires, Argentina, and is essentially the Boivin-type fraction of the abortus and melitensis species (Barrera Oro, Peroshi & Molinelli-unpublished data, 1957). Four units of complement were employed in the test, which was read at its conclusion in terms of 100% haemolysis. The method used here is very similar to the procedure of Renoux (1957), who uses two units of complement but employs either a heated cell suspension or a soluble antigen obtained by freezing and thawing brucellae. Flocculation test This test was performed according to the method of Hunter & Colbert (1956) with antigen supplied by Dr C. A. Hunter, Kansas State Laboratories of Public Health. Milk ring test The phenyltetrazolium reagent (Bendtsen, 1954) and the haematoxylin reagent (Agricultural Research Service, USDA) were used in the amounts recommended, i.e., 2 drops per 2 ml of milk sample. The tubes were incubated at 37°C for various periods before being read. Cutaneous hypersensitivity The non-antigenic preparation described by Ottosen & Plum (1949, 1952) was employed. An intrapalpebral injection of 0.1 ml of this prepara- tion was given and the reaction was read at 24 and 48 hours by measurement of the increase in skin thickness. Blood culture Approximately 5 ml of venous blood was inoculated directly into 75 ml of Albimi broth, shaken vigorously and incubated at 37°C. At weekly intervals subcultures were made on to Albimi agar. The broth cultures were incubated for at least 3-4 weeks before being discarded as negative. All cultures were identified as being Br. melitensis or not on the basis of colonial appearance on Albimi agar, and slide agglutination with monospecific, absorbed sera, tube agglutination with specific sera, and bacteriostatic dye tests. 1036 S. S. ELBERG Milk culture Milk was tested within 4-5 hours of collection by plating the cream and sediment obtained after centrifugation of the sample at 6000 revolutions per minute in a refrigerated machine. The specimens were plated directly on to Renoux's modification (1954b) of the Kuzdas & Morse (1953) medium and on to Albimi agar. The plates were incubated for 10 days before being discarded as negative. Autopsy procedure The animals were sacrificed by an intravenous injection of 1 ml of "Buthanasia ". The following tissues and organs were examined for the presence of the challenge strain of Br. melitensis by grinding portions of the tissue or organ in sand, suspending in saline, and culturing on Renoux's modification of the Kuzdas & Morse inhibitory medium: right and left suprascapular, hepatic, iliac, right and left supramammary, right and left precrural lymph-nodes, liver, lung, mammary gland, spleen, uterus and heart blood. AU cultures suspected ofbeing Br. melitensis were identified by the series of tests described above. Selection of animals Out of 176 goats available, 96 were found to be negative to the agglutina- tion and cutaneous hypersensitivity tests on at least 2-4 occasions before immunization. The animals were obtained locally in the Province ofCordoba and were 11-18 months old when used. A number of younger animals (3-4 months old) were also vaccinated, but a severe winter and feeding problems eliminated this group almost entirely by the time of autopsy, leaving 54 adult animals for the final analysis. Procedure The animals were injected with 1 x 109 cells of the vaccine strain subcutaneously in the left suprascapular area. After 12-14 weeks groups of animals were challenged with graded doses of a virulent Br. melitensis strain in the right suprascapular area. At the same time non-immunized animals were also infected with graded doses of the challenge strain. During the pre-challenge period the immunized animals were bled at weekly intervals for blood culture and serological tests. Blood samples were taken from the control-group animals as well, after the challenge infection had been administered. Animals were classified as infected on the basis of a single colony of brucellae isolated from a single lymph-node. Results Immunity studies The number of infected goats in each challenge group is presented in Tables 1 and 2.