Strain 481 is a peculiar one among the collection of 49 strains. It is the only strain in the collection which agglutinates spontaneously under the conditions of the methods employed in this study for the simple agglutination test. Owing to the spontaneous agglutination of serum 481, the data showing absorption of agglutinins from that serum are omitted. The data obtained by absorbing agglutinins with this strain from the various type serums show that it is rather distantly related to the important types represented by strains 426 and 104, and it absorbed no agglutinins from serum 451. Strain 481 was of porcine origin. It was the only strain, belonging to its serological group.

Strain 480 also was the only one found belonging to its serological group. It is a European strain of bovine origin. It forms a connecting link between the serological groups represented by strains 481 and 426.

Strain 451 represents an important group which includes strains of human, caprine, bovine, and equine origins. Three of the four American strains of human origin available for this study belonged to this serological group. It includes strains from four different animal species, isolated in the United States, Europe, and North Africa. Serologically it forms a connecting link between the groups represented by strains 426 and 104.

Strain 101 also represents an important, though a small group. The three strains of this group are of a peculiar morphology, with a majority of coccoid cells and comparatively few elongated forms. Their history is discussed in the preceding paper, in which it is pointed out that it is probable that Bruce was working with strains of this group when he gave the generic name "Micrococcus" to his organism.

TABLE VI.-Cross agglutinin reactions between the various serological groups of Brucella melitensis.

The figures give the highest dilutions in which a positive reaction (75 per cent or more sedimentation) occurred.

The cross simple agglutinin reactions between the six serological groups of melitensis represented by strains 480, 426, 457, 427, 451, and 104 are given in Table VI. In general it may be stated that an unknown strain can not be identified with any one of these six

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serological groups by the simple agglutinin test. The cross agglutination between the group represented by strain 104 and the remaining strains was generally lower than agglutination of the various strains in their homologous serums. But this difference in agglutinin response is not enough to differentiate the serological group when the simple agglutinin test is being carried out for diagnostic purposes with an unknown strain, or with an unknown serum.

It is pointed out in the preceding paper that these closely related serological groups belong to a single species, Brucella melitensis, and that the serological groups may be designated as varieties. The group represented by strain 426 may be designated variety abortus; the group represented by strain 451, may be designated variety melitensis A; and the group represented by strain 104 may be designated variety melitensis B.

As Table IV and Chart 1 show, the group represented by variety melitensis A stands between varieties abortus and melitensis B in serological relationship. That the simple agglutinin reaction is useless in distinguishing the abortus and melitensis A varieties has been demonstrated in a practical way a number of times when serums from human cases of melitensis infection were tested with antigens of both the homologous and heterologous serological groups. These data are brought together in Table VII. It may be noted from this table that an antigen of the heterologous serological group agglutinated in a higher titer of the serum than the antigen of the homologous group in two of the five cases.

1

TABLE VII.-Cross agglutination of human serums in cases of melitensis infection with antigens of the homologous and heterologous serological groups.

a Strain 455, belonging to the same serological group as strain 426, was used in these tests. The infecting strain was not isolated, but its serological grouping was determined by absorption of agglutinins from the serum with the various type antigens.

After the 49 strains included in this study had been classified, a small collection of 9 strains of human and caprine origins was received from Doctor Burnet, of the Pasteur Institute at Tunis. None of the 9 strains was identical with the types already established. Judged by their capacity for absorbing agglutinins from the type serums, they are a heterogeneous lot, including several new serological

types, most of which are more closely related to the group represented by strain 104 (melitensis B) than to the other serological groups. They are unlike the abortus and melitensis varieties in agglutinating in nonspecific serums and they have a greater tendency to spontaneous agglutination in salt solution. Compared with the melitensis strains previously studied, the Tunis strains are poor anti-body producers. They agglutinate well in serum 104, many, but not all, of the strains reacting in as high titer as the homologous strain. They agglutinate poorly in serum 426 (abortus variety) and serum 451 (melitensis A variety).

Previous to 1912 there was a considerable doubt cast upon the specificity of the agglutinin test for Br. melitensis infections, because inconsistencies in its application, occurred. The discovery by Nègre and Raynaud of a strain which failed to agglutinate, or agglutinated only slightly, in melitensis serums. explained the inconsistencies and reestablished the test on a firm basis. They designated as paramelitensis those strains which were similar to melitensis in morphological, cultural, and biochemical behavior, but failed to agglutinate or agglutinated slightly in melitensis serum. Absorption of agglutinins by paramelitensis from melitensis serum was only partial, whereas absorption of the same serum by four melitensis strains was complete. Feusier and Meyer found that paramelitensis produced lower titers of agglutinins in rabbits than did melitensis. Smith states that paramelitensis is more easily agglutinable in nonspecific serums than is melitensis, and that it is more autoagglutinable. The Tunis strains agree in these respects with the descriptions of the paramelitensis of Nègre and Raynaud.

DISCUSSION.

Bassett

It is unfortunate that only one of the 49 strains included in this study was of caprine origin. Presumably in a district where human cases of Malta fever occur, due to consumption of infected goat's milk, the human and caprine strains are of the same serological grouping. The following facts are the only experimental evidence at hand to substantiate that assumption. A human and a caprine strain received from Milan, Italy, both belonged to the variety melitensis A. Agglutinin absorption tests with a goat serum of good titer from the Phoenix, Ariz., district indicate that the infecting organism belonged to variety melitensis A, as did the human strains 451 and 506, from cases which contracted their infection in that district.

The data presented in this paper confirm the accumulating evidence that the organism causing contagious abortion in cattle and swine belongs to the same species as the organism causing Malta fever in

man. Although one serological group is characteristically of bovine and porcine origin, and another group is characteristically of human (and caprine (?) ) origin, it is apparent (Table III) that the serological groups are not limited to the host species for which they are characteristic.

The results of the study of these 49 strains confirm the conclusions. of Feusier and Meyer that their groups 1 and 2 are very closely related, and their group 3 (melitensis A) is sharply defined from groups 1 and 2. The results of this study are at variance with Feusier and Meyer's in that only 2 of the 49 strains were found belonging to their group 2 (a subvariety of abortus), whereas they classified 9 of their 14 strains in that serological group.

SUMMARY.

The agglutinin absorption tests with 49 strains of Brucella melitensis have shown that

(1) This species may be differentiated into at least seven serological groups. Four of these groups included only one or two strains each, and were relatively unimportant.

(2) The majority of bovine and porcine strains fell into one large group (30 strains), which is designed variety abortus. Two strains of human origin were of this variety. Two of the small serological groups are so closely related to this one that they should be considered as subvarieties. One of these includes only one strain of bovine origin; the other includes two strains, both of human origin.

(3) Another important group includes strains of human, bovine, caprine, and equine origins (11 strains). It is designated variety melitensis A.

(4) A third group of three strains characterized morphologically by a predominance of coccoid cells assumes importance from the evidence that this was the variety that Bruce was working with when he named his newly discovered organism "Micrococcus melitensis." It is designated variety melitensis B.

Simple agglutination tests can not differentiate between varieties abortus and melitensis A. These two varieties can be differentiated from melitensis B by the simple agglutination test only when the titer of the serum used is accurately known for the several varieties.

REFERENCES.

American Public Health Association (1920): Standard methods for the examination of water and sewage. 4th ed. Boston. p. 4.

Bassett-Smith, P. W. (1921): Paramelitensis infection in man and animals. J. Trop. Med. and Hyg., 24, 53-54.