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STUDIES ON BRUCELLA MELITENSIS

I. THE SEROLOGIC CLASSIFICATION OF STRAINS FROM

HUMAN, BOVINE, CAPRINE, PORCINE, AND EQUINE
SOURCES

By ALICE C. Evans, Associate Bacteriologist, Hygienic Laboratory, United

States Public Health Service

REVIEW OF THE LITERATURE The observation that the causal organism of Malta fever and that of contagious abortion in cattle are closely related in their morphological, cultural, biochemic, serologic, and pathogenic characteristics (Evans, 1918) was confirmed by Meyer and his associates in this country, by Zeller and by Jaffé in Germany, by Skarić in Austria, by Mazzi, by Auricchio, and by Polettini in Italy, by Hoeden in the Netherlands, by Khaled in Egypt, by Burnet in Tunisia, and by Futamura in Japan. Fleischner, Meyer, and Shaw found, further, that the test for cutaneous hypersensitiveness, which they regard as highly specific, can not differentiate experimental infections in guinea pigs caused by Brucella melitensis varieties abortus and melitensis. Burnet subjected four Malta fever patients to the intradermal test with three Br, melitensis antigens, one prepared with the melitensis variety and two with the abortus variety-a bovine and a porcine strain, respectively. The three reactions were positive at the same time in all four cases, and in all four cases the porcine strain provoked the strongest reaction.

Fleischner, Vecki, Shaw, and Meyer compared the pathogenicity for monkeys of strains of Br. melitensis from human and from bovine sources. By feeding large doses of virulent strains of the abortus variety they were able to infect monkeys as demonstrated by isolation of the organism from the viscera and heart blood at necropsy. The melitensis variety was far more invasive, however, causing infection in comparatively small doses.

Meyer and his associates also studied the pathogenicity of the abortus and melitensis varieties of Br. melitensis for guinea pigs. They found the abortus variety slightly more invasive and virulent,

1 Manuscript submitted for publication May 18, 1925. * See the following article for a discussion of the nomenclature of this bacterial species. 50587-25—2

(1)

as a rule, than the melitensis variety. The gross anatomic and the histologic changes were so nearly alike for the two infections, however, that they could sometimes be differentiated only by careful serologic cross absorption tests with the isolated organisms. Jaffé also found that the inflammatory changes in the case of Br. melitensis varieties abortus and melitensis infections in guinea pigs were qualitatively alike, with more pronounced changes in the case of the abortus infections. Burnet also states that he is of the impression that the abortus variety is more virulent than the melitensis variety for guinea pigs. On the contrary, Khaled found the melitensis variety much more virulent than the abortus variety for guinea pigs.

Huddleson found that the tissue changes produced in guinea pigs by virulent strains of the melitensis variety could not be distinguished from those changes produced by virulent strains of the abortus variety. Certain strains of both varieties failed, however, to produce the characteristic lesions in guinea pigs. The author attributes the lack of virulence to long cultivation in an artificial medium. It is well recognized that pathogenic bacteria lose their virulence when grown on artificial media, and this loss in virulence has been commonly noted in the organism of contagious abortion. Hence a fair comparison of the virulence of the varieties of Br. melitensis can not be made with strains whose histories differ greatly.

Burnet and de Lagoanère experimented with white rats, gray rats, white mice, and gray mice, to determine whether inoculation of these animals would distinguish between the abortus and melitensis varieties. The tests showed no difference in the pathogenicity of the two varieties.

Khaled immunized a monkey by treating with the abortus variety and found a mild reaction to an infecting dose of the melitensis variety as compared with an untreated control monkey. In another experiment reported in a later publication, Khaled immunized a monkey with killed cultures of abortus. This monkey and a control were then given a dose of living melitensis. The immunized monkey lost slightly in weight, but soon regained normal weight, whereas the control lost about 350 grams. After three months both monkeys were autopsied. Br. melitensis was obtained from the blood and spleen of the control, but it could not be recovered from the tissues of the immunized monkey.

Burnet also immunized two monkeys with abortus, one with a living, the other with a killed culture. Both were later inoculated with living melitensis, without effect.

Khaled immunized two goats by treatments with living abortus, and found them protected against experimental infection with the melitensis variety, as compared with a control goat which had not been immunized.

Mazzi treated one group of white rats with anti-abortus serum and another group with anti-melitensis serum. Twenty-four hours later he injected them with lethal doses of melitensis or abortus. He found that the anti-abortus serum protected the rats against melitensis as well as against abortus, and the anti-melitensis serum protected against abortus as well as against melitensis.

There have been several recent reports of the use of abortus as vaccines for human subjects. Burnet inoculated two human subjects with living cultures of an old laboratory strain of abortus of porcine origin. Subsequent inoculation with a virulent caprine strain of mėlitensis resulted in a mild disability in one subject. The other subject suffered no ill effects from the experiment. Khaled treated three cases of human Malta fever with killed abortus vaccine and reported favorable results. Auricchio treated 15 cases with abortus vaccine and reported favorable results in every case.

Aside from these experimental evidences of a close relationship between the bovine and caprine strains of Br. melitensis, the natural course of the disease in the two species of animals is conspicuouly similar. Both cattle and goats may harbor the Br. melitensis for years without evident impairment of health other than abortion; both species of animals may eliminate the organism in their milk for long periods, although apparently in good health. In the cattle disease attention has been directed to the abortion of the fetus because of its great economic importance. On the other hand, in the goat disease attention was directed in the past chiefly to the infectiousness of the milk, and the abortion of the fetus was not so much emphasized, although it has long been recognized as a symptom of infection. In 1911 Dubois called attention to abortion as the chief symptom of the disease in goats and sheep, and Holt and Reynolds recently reported that along the Mexican border there is occasionally 50 per cent loss from abortion in some herds of goats.

Feusier and Meyer made a study of the agglutinin absorption reactions of 1 bovine and 11 human strains of Br. melitensis and 2 strains of paramelitensis. The strains fell into groups as follows:

Group 1: One human strain of Br. melitensis, and the one

bovine strain.
Group 2: Nine human strains of Br. melitensis.
Group 3: One human strain of Br. melitensis.

Group 4: Two strains of Br. paramelitensis. Groups 1 and 2 were so closely related that they could not be differentiated by simple agglutination tests, although certain differences were manifest in agglutinin absorption tests. Thirty-two strains isolated from aborted fetuses or pathologic discharges or milk of cattle and hogs in this country or in England all fell into

group 1, according to the tests that were made. The authors state, however, that the complete absorption technique was carried out with only one strain of the abortus variety.

A review of the literature, therefore, leaves no question about the close relationship between strains of Br. melitensis from bovine and human sources.

SCOPE OF THIS STUDY

The epidemic of Malta fever which occurred in Arizona in 1922 (reported by Lake), and a few sporadic cases which have come to our attention, have renewed an interest in the causal organism of this disease. Since the agglutinin absorbing properties constitute the only recognized point of distinction between the strains of Br. melitensis, it appeared important that a larger series of human strains than that of Feusier and Meyer should be serologically classified and compared with caprine strains and with the organism causing contagious abortion in cattle and other species of domestic animals.

Accordingly, a collection of strains has been made. The sources of the strains and the date of isolation, as far as the information could be obtaired, are recorded in tabular form (Table I). The writer is indebted to the many investigators whose names appear in the table for generous response when requests for cultures were made. Altogether 68 strains were received and serologically classified.

TABLE I.--History of the strains
STRAINS FROM HUMAN SOURCES

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102 (?).

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103 (?)...

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426' (?).

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Hygienic Laboratory collection.

Obtained from England in 1907.
Labeled "M. melitensis," Dr.

Annett.
(?).

Hygienic Laboratory collection.

Ohtained from Royal Army
Medical Corps, London, Eng-
land, in 1908.
Hygienic Laboratory collection.

Obtained from U.S. Naval Medi

cal School in 1909. (?) Austria (?). Dr. K. F. Meyer. Originally from

Royal Army Medical Corps, Lon.

don, England.
(?).
Sicily (?).

Dr. K. F. Meyer. Obtained from

Dr. Guido sar, Catania, Sicily. (?). Algeria (?) Dr. K, F. Meyer, Obtained from

Dr. E. Sergent, Institut Pasteur

d'Algerie, Algiers. September, 1922... Phoenix, Ariz....Dr. G. C. Lake. November, 1922... Baltimore, Md... Dr. H. L. Amoss, Johns Hopkins

427 (?). 428 (?).

| 451

Blood. 455

do

University Hospital, Baltimore,

Md.
(?)
(?)...

Army Medical School, Washington,

D. C. Obtained from New York

City.
1911.
Texas...

Army Medical School, Washington,

D, C.

461 (?)

462 (?)

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463 (?).

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464 Blood...

489 (?)..

505 (?)

506 Blood..

(?).

Army Medical School, Washington,

D. c. Obtained from Parke,
Davis & Co., Detroit, Mich.

Originally from Kral's collection.
1896.
London, Eng... - Society of American Bacteriolo-

gists' collection. Labeled "Mi.

crococcus melitensis 33."
(?).
(?).

H. K. Mulford Co. culture No. 545.

Obtained from Kral's Museum

in 1913. (?). Italy (?)..... Dr. C. Gorini, Laboratorio di Bat

teriologia, Scuola Superiore Agri

coltura di Milano. May (?), 1923.... New York City... Dr. L. W. Famulener, St. Luke's

Hospital, New York City. Infection was contracted in Phoe

nix, Ariz.
1920..
Tunisia.... Dr. Ét. Burnet, Institut Pasteur

de Tunis.
May, 1921
...do

Do.
April, 1923
--do

Do.
1920.

.do.

Do.
May, 1923
Malta..

Public:Health Department, Malta. 1924.

Sioux Falls, S. Dr. D. A. Gregory, Sioux Falls,
Dak.

S. Dak.
July, 1924. New Haven, Conn. Dr. Kirby A. Martin, New Haven,

Conn.
February, 1925... Ithaca, N. Y... Dr. Charles M. Carpenter, New

York State Veterinary College,
Ithaca, N. Y.

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STRAINS FROM BOVINE SOURCES

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456 Cow's fetus... September, 1917... Laurel, Md.-- Bureau of Animal Industry, U, S.

Department of Agriculture,

Washington, D.O. 457 (?). Prior to Decem- (?)-

Dr. B. A. Beach, College of Agriber, 1918.

culture, Madison, Wis. Labeled

“Wis. W." 458 (?)...

(?).

Dr. B. A. Beach, College of Agri

culture, Madison, Wis. Labeled

“Wis. M." 459 Cow's fetus... January, 1917.-.-- Gambrills, Md... Bureau of Animal Industry, U. S.

Department of Agriculture,

Washington, D. C. 460 (1) January, 1920...- Michigan (?)..---- Dr. I. F. Huddleson, Michigan

Agricultural College, East Lan

sing, Mich., "No. 200." 465 Stomach of abort. December, 1922.-- Minnesota (?). Dr. O. P. Fitch, University of Mined fetus.

nesota. 466 Colon of aborted January, 1923 New York State(?) Dr. W. A. Hagan, Cornell Univerfetus.

sity, Ithaca, N. Y. 467 Duodenal contents 1919..

Connecticut (?)... Dr. Leo F. Rettger, Yale Univerof prematurely

sity, New Haven, Conn. Laborn calf.

beled "St. 4." 468 .do

1920.

..do

Dr. Leo F. Rettger, Yale Univer

sity, New Haven, Conn. La

beled “St. 6." 474 Aborted call... February, 1922... Gilbert, Iowa ... Dr. S. H. NcNutt, Iowa State Col

lege, Ames, Iowa, Labeled

"Orawford."" 476 | Aborted fetus..--- December, 1917.-- Beltsville, Md... Bureau of Animal Industry, U. S.

Department of Agriculture. 477 Uterine exudate..- (?)..--.

Province Zuid, State Serum Institute, Rotterdam,
Holland.

Holland. 478 Aborted call... (?)...

Province Zeeland,

Holland. 479 --...do....

(?).

Province Gelder Do.

land, Holland. 480 (?).

(?)..---
(?)--

Prof. K. Suffle, Universität Mün

chen, Germany. 485 (?).

(?).-------- Zurich, Switzer- Dr. G. Sobernheim, Institute zur land.

Erforschung der Infektionskrank

heiten, Berne, Switzerland. 497 (?). Prior to April, (?).

Dr. Edgar B. Carter, Swan-Myers 1916.

Co., Indianapolis, Ind. Labeled "B. abortus 101.1."

Do.

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