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smears of the various serologic groups are examined directly under the miscroscope.
Ít appears probable that Bruce was working with strains of a serologic type in which coccoid forms dominate the microscopic appearance to a greater extent than in other serologic types. It is the writer's opinion, however, that these strains should be considered as belonging to the same species as those strains in which the bacillary forms are more predominant, for the strains of slightly different morphologies are identical in cultural and biochemic reactions, and they can not be distinguished by the simple agglutination reactions.
In 1918 the writer made the observation that there is a very close relationship between the Malta fever organism and the so-called “ Bacillus abortus” which Bang, in 1897, had established as the cause of contagious abortion in cattle. The generic name “Bacillus," which formerly had been applied promiscuously to all rod forms of bacteria, was no longer in conformity with the nomenclature adopted by the Society of American Bacteriologists, since the Committee of the Society of American Bacteriologists on Characterization and Classification of Bacterial Types, in its 1917 report, restricted the generic name “Bacillus” to aerobic spore-bearing rods. The nonspore-bearing pathogenic rod forms were classified in the genus “Bacterium.” Thus “ Bacillus abortus” became " Bacterium abortus," ! and the closely related “Mierococcus melitensis " became * Bacterium melitensis." Following the writer's observations that the causal agent of Malta fever in man and that of contagious abortion in cattle are closely related, and that the so-called “Bacillus bronchisepticus "—the cause of distemper in dogs and of a similar disease in other animals-resembles them morphologically, culturally, and biochemically, the committee suggested (1920) that if these observations were confirmed the mentioned organisms should probably constitute a new genus, because they differ so widely from the type species of the g nus Bacterium.
After Fleischner, Meyer, and Shaw had confirmed the observation that the Malta fever and contagious abortion organisms are closely related, Meyer and Shaw (1920) proposed the generic name “Brucella," in the family Bacteriaceae, to include the Malta fever and contagious abortion organisms. That name has met with general approval and has been used by foreign investigators (Khaled, Archibald, Béguet, Hoeden, etc.).
* In its final report on the families and genera of the bacteria (J. Bact. 1920, 5: 19).229), the committee changed the specific name "abortus " to "abortum," presumably to have the ending agree with Bacterium. This was an error, for abortus is not an adjective, but a Latin noun in the genitive of the third declension. Hence its ending is independent of the ending of the generic name.
Meyer and Shaw did not, however, give a generic diagnosis for the genus Brucella, nor did they consider other species besides the melitensis-abortus group which would logically belong to the new genus. They were apparently unaware that Castellani and Chalmers had already described a newly created genus, “Alcaligenes," which, according to its definition, would include the melitensisabortus group. The definition is as follows: "Bacillaceae growing well on ordinary laboratory media; not forming endospores; aerobes, and often faculative anaerobes; without fluorescence, pigment formation, or gelatin liquefaction; without polar staining; Gramnegative, without a capsule
Milk not clotted; glucose and lactose not fermented." The type species of the genus “Alcaligenes” as established by “ original designation,” is A. fecalis, a common intestinal saprophyte. Castellani and Chalmers left the “Micrococcus melitensis” unclassified generically—“ Incertae sedes ”-because they were doubtful as to whether it should be considered a coccus or a rod form.
Bacteriological nomenclature is passing through an experimental, transitional stage, and criteria which should serve for valid specific or generic distinctions have not been established in detail. The characteristics which are available at present for bacterial classification are few; they are more or less variable according to varying conditions; and they are not well correlated. Hence comes the difficulty in classifying bacteria into a system comparable with that of the higher organisms. It appears that, in many cases, distinction must be made by means of the sum total of differences, rather than by differences in particular 'stable characteristics, such as obtains in the classification of higher organisms.
If melitensis is considered cogeneric with fecalis, then the Law of Priority demands the acceptance of the generic name Acaligenes. In his recent “Manual of Determinative Bacteriology” Bergey placed melitensis in the genus Alcaligenes, and Orpen is of the opinion that melitensis and fecalis are closely enough related to be included in the same genus. The melitensis-abortus group has been referred to occasionally elsewhere in recent literature under the generic name Alcaligenes.
If, however, the differences between the species melitensis and fecalis are sufficient for generic distinction, the valid name of the genus to which melitensis belongs is Brucella.
Brucella. The writer is of the opinion that a generic distinction should be made between the organisms under discussion, which are characteristically invaders of the tissues of animals, and the type species of Alcaligenes, which is characteristically a saprophyte. It may be noted here that Smith has shown that “Bacillus abortus " invades the chorionic epithelial
cells, which indicates a high degree of parasitism, as compared with other pathogenic bacteria. Localization and multiplication of bacteria within cells not having phagocytic functions has been demonstrated in only a few diseases.
Morphologically, also, there is a marked difference between melitensis and fecalis, the former being distinctly smaller than the latter. Smears of two-day agar cultures of Br. melitensis stained with carbol fuchsin show minute organisms about 0.5 micron wide and from 0.5 to 2 microns long. Smears of Alcaligines fecalis prepared in the same manner show organisms 0.8 to 1 micron in width and commonly 3 microns long, with occasional cells 10 or 20 or even more microns long
A further distinction is in the reduction of nitrates to nitrites by fecalis, whereas melitensis fails in this reaction.
There is given herewith a general description of melitensis, •the type species of the genus Brucella:
Minute rods with many coccoid cells; (the cells of 2-day cultures grown on the surface of plain agar and stained with carbol fuchsin appear about 0.5 of a micron wide and 0.5 to 2 microns long) ; not forming endospores; nonmotile; aerobic, or preferring a slightly reduced, partial pressure of oxygen; without gelatin liquefaction; Gram-negative; parasitic, invading animal tissues; neither gas nor acid production from the carbohydrates.
Other species, which vary somewhat from the foregoing description, will logically be allocated to the genus Brucella. This genus should include a variety of small rod-forms commonly present in freshly drawn cow's milk. These forms were described in an earlier publication (Evans, 1918) as bacteria related to "Bacterium abortus." Some of them vary from the typical Brucella in the production of a slight amount of acid from the carbohydrates. The genus Brucella should also include the species bronchisepticus, which varies from the typical Brucella in being motile. A number of other species should also probably be classified in the genus Brucella.
Meyer and his associates have continued the use of the abbreviation “B.” for the generic designation of melitensis and abortus. Since that is the accepted abbreviation for the genus Bacillus, there should be some other for the genus Brucella. The abbreviation Br. is proposed for that genus.
But the point may be made here that whenever a generic name is referred to in any paper it should be printed in full the first time it is cited in the article.
Every investigator since 1918 who has compared the Malta fever and the contagious abortion organisms has found a close relationship between them. The literature on this subject is reviewed in the preceding article. The accumulating evidence of the close relationship between the strains of bovine and human origin has culminated
in the conclusion by Burnet that melitensis and abortus are not distinct bacteriological species, but merely distinct serologic varieties or subspecies of one and the same species. All the literature on the subject leads to this conclusion, in which the writer concurs. Observing priority of publication as determining the nomenclature, we must adopt the name melitensis as the specific name for the melitensis-abortus group.
In the preceding paper there is presented a considerable amount of serologic data which show that strains of human, bovine, porcine, caprine, and equine origin can not be distinguished by the simple agglutination test. By the agglutinin absorption test these strains fall into distinct serologic types which may be considered as varieties or subspecies if the entire group is considered as a single species. The proposed designations for the serologic groups of varieties are discussed in the preceding paper, together with their descriptions.
Any argument which might be presented in favor of a species distinction between the abortus and the melitensis serologic groups would apply also to the separation into distinct species of the serologic groups designated varieties melitensis A, melitensis B, and paramelitensis in the preceding paper, and the single strains 481 and 480 discussed in that paper would represent two more distinct species. From the point of view of the biologist there is no justification for such a division of the abortus-melitensis group into many species. Jordan and Kellogg express the generally accepted view of biologists as follows:
A series of fully intergrading forms, however, varied at the extremes, is usually regarded as forming a distinct species * *. If we find actual intergradation, the occurrence of specimens intermediate in structure, the term subspecies is commonly used for each of the recognizable groups thus connected.
In the classification of bacteria, where distinctions are made by means of biochemic or serologic reactions, as well as by means of morphology, a series of fully intergrading reactions should be regarded as a species, to conform to the generally accepted definition of the term. Chart I of the preceding paper shows that the serologic relationships between the eight melitensis-abortus groups unite them into an intergrading series. Hence they should be regarded as a single species, for there is no characteristic other than their serologic behavior by which they may be differentiated.
The writer is indebted to Dr. C. W. Stiles, of the Hygienic Laboratory, for advice concerning the principles of Linnean nomenclature.
STUDIES ON BRUCELLA MELITENSIS
III. EXPERIMENTAL ABORTION IN A COW PRODUCED BY
INOCULATION WITH A HUMAN STRAIN 1
By ALICE C. Evans, Associate Bacteriologist, United States Public Health
In the first paper of this bulletin a review is given of the comparative studies which have shown the close relationship between the organism causing contagious abortion in cattle and other domestic animals and that causing Malta fever in man and in goats. Further data are recorded in that paper which show that aside from differences in degrees of virulence for man and monkeys the distinction between the strains which are characteristically bovine or human is apparent only as a slight difference in their agglutinin-absorbing properties. It is also shown that the bovine type (variety abortus) of Brucella melitensis may infect man and that the human type (variety melitensis A) may infect cattle. This information suggested that experimental inoculation of a pregnant heifer with a virulent strain of human or of caprine origin should result in abortion, as is the case when a susceptible pregnant cow is inoculated with a virulent strain of the abortus variety.
Experiment 1.-On February 9, 1923, a pregnant heifer, No. 981 A, was inoculated intravenously with Br. melitensis strain 451. (See Tables 1 and 3 and Chart I of the first paper of this bulletin for information about the strains used in these inoculation experiments.) Strain 451 had been isolated a few months previously from a human case of Malta fever contracted from goat's milk. This strain was believed to be of high virulence. The dose was the growth from a 24-hour culture on a serum glucose agar slope suspended in 10 cubic centimeters of physiological saline solution. On March 26, 1923, the heifer aborted a fetus of about five and one-half months. Br. melitensis was recovered from the stomach contents, from the contents of the small intestine, from the peritoneal fluid of the fetus, and from the colostrum.
The recovered strain was shown to be serologically identical with the strain used for inoculation. This strain of the melitensis A variety is unmistakably distinguishable from the typical bovine variety of Br. melitensis by the agglutinin-absorption test. The data for the absorption tests which identified the recovered strain are given in Table 1. The serum used for these tests was a rabbit serum prepared by inoculation with strain 451. The strain recovered from heifer 981 A is designated 451 A. Strains 426 and 468 in the table belong to the abortus variety of Br. melitensis. Strain 426 is of human origin,
1 The writer is indebted to Dr. E. C. Schroeder and Dr. W. E. Cotton, of the Bureau of Animal Industry Experiment Station, United States Department of Agriculture, for providing the animals used in carrying out the experiments and for the care of the animals. 50587-23—4