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and strain 468 is of bovine origin. Details of the technic employed in making absorption tests were described in the first paper of this bulletin. In these tests the absorptions were made with 5.7 c. c. of antigen of a density equivalent to 20,000 parts per million of the silica standard, which was centrifugalized, 2.85 c. c. of the clear fluid removed, and 0.15 c. c. of serum added.

TABLE 1.-Identification of the strain recovered from heifer 981A by comparison

of its agglutinin absorption properties with those of the inoculating strain and with those of strains belonging to the abortus variety

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14, complete sedimentation; 3, supernatant turbidity as in a control tube containing 25 per cent as much antigen as in the tubes in which the test was carried out;

2, supernatant turbidity as in a control tube containing 50 per cent of the antigen; 1, supernatant turbidity as in a control tube containing 75 per cent of the antigen.

The simple agglutination test was with the homologous antigen.

The agglutinin titers of the cow's serum taken before inoculation and at subsequent intervals of about two weeks are given in Table 2. Tests for agglutinin content were made with the inoculating strain and with the bovine strain 468 of the abortus variety.

TABLE 2.-Agglutination of Br, melitensis antigen in the serum of heifer 981A

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The presence of agglutinins in low dilutions of the serum previous to inoculation is not considered indicative of an infection, because cow's serum occasionally contains agglutinins for Br. melitensis in small quantity. The simultaneous rise in agglutinins for antigens of the melitensis A and the abortus varieties is in agreement with the findings reported in the first paper of this bulletin.

Experiment 2.-Another pregnant heifer, No. 1014A, was inoculated on the same date, and in the same manner as heifer 981A, but the inoculating strain, 427, of human origin, was more closely related to the abortus variety than the strain used in the experiment described above. To our knowledge strain 427 had been under artificial cultivation for several years although the exact date of its isolation was unknown. It has been commonly observed that the

virulence of Br. melitensis is impaired under artificial cultivation, hence the pathogenicity of strain 427 was uncertain. On May 7, 1923, heifer 1014A aborted. A few hours later the stomach contents, intestinal contents, and liver of the fetus were planted on glucose agar plates and slopes. All the cultures developed abundant colonies in practically pure culture. The recovered strain was not 427, however, but an organism which grew faintly on the agar slopes and in broth culture and produced acid in glucose broth.

The agglutinating titer of the serum from heifer 1014A for Br. melitensis before inoculation and at intervals thereafter, and the titer of the colostrum on the day of the abortion are given in Table 3.

TABLE 3.--Agglutination of Br. melitensis antigen in the serum and colostrum

of heifer 10144

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See footnote to Table 1 for the significance of the figures.
? The opacity of the colostrum interfered with exact readings in the lower dilutions.
The simple agglutination test was with the inoculating strain No. 427.

The rise in the agglutinin titer of the serum from heifer 1014A following the inoculation and the occurrence of abortion about three months later suggest that the inoculating strain produced an active infection. The results are confused, however, by the overgrowth of all cultures with an organism which was not Br. melitensis. The part which this unknown organism may have played in causing the abortion is problematical.

Conclusion.—Experimental abortion was induced in a pregnant heifer by inoculation with a culture of Br. melitensis, variety melitensis A, from a human case of Malta fever, and the inoculating strain was recovered from the fetus and the colostrum.

STUDIES ON BRUCELLA MELITENSIS

IV. CATTLE AS A SOURCE OF HUMAN INFECTION

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

States Public Health Service

Ever since it has been known that the causal organism of infectious abortion in cattle is of common occurrence in cow's milk, the question has been raised from time to time as to what effect this germ may have on human health. The question became more emphatic when it was established that Bang's Bacillus abortusand the “Micrococcus melitensisof Bruce are so closely related that strains from bovine and caprine sources are now considered to belong to the same bacterial species.

A series of tests carried out with Brucella melitensis antigen and serums from patients suffering from various kinds of diseases furnished data bearing on the infectiousness of the abortus variety of Br. melitensis for man, although the study was undertaken primarily with a somewhat different problem in view.

In testing human serums from suspected Malta fever cases, the question came up in the Hygienic Laboratory as to how high a titer of agglutinins specific to Br. melitensis was necessary to lead to the conclusion that there was actually an infection with that organism. To aid in forming a definite opinion it was advisable to accumulate some data on the agglutinin response of Br. melitensis in serums from cases in which Malta fever was not suspected. A number of positive reactions were obtained, which led to the consideration of the infectiousness of the bovine type of the organism for man.

A review of the literature dealing with the prevalence of Br. melitensis in cow's milk and the infectiousness of the bovine strains for man is a logical preface to a study of the agglutinin reactions of human serums with the melitensis antigen. There are to be found in the literature a few reports of cases of Malta fever which could not be traced to infection from goats and for which no other source of infection could be established. As it now appears possible that the source of infection in some of those cases may have been cow's milk, the literature concerning them is included in the review.

In an attempt to interpret the significance of the positive reactions obtained a comparison should be made with the agglutinin titers which are considered indicative of Br. melitensis infections in regions where Malta fever is endemic. A brief review of the literature relating to this subject is therefore included.

REVIEW OF THE LITERATURE

CASES OF MALTA FEVER WHICH COULD NOT BE TRACED TO INFECTION FROM

GOATS

Birt and Lamb reported a case of Malta fever which they conclude was contracted either in Plymouth or in London, although no definite source of infection could be traced.

In 1905 Craig reported a case of Malta fever which was the first on record originating in the United States. The patient was a hospital nurse. There were no cases of Malta fever in the hospital at the time of her attack, but she had previously been attending sick soldiers in a hospital in Washington, D. C., and Craig thought it possible that she had acquired Malta fever in that way. No other possible source of her infection was suggested. In the paper in which Craig reported the above-mentioned case, together with other cases in which the disease was contracted outside of the United States, he makes the following statement :

I am convinced that a careful study, by use of the Widal test and the agglutination reaction with Micrococcus melitensis, of many of the cases of obscure continued fevers which are prevalent in this country will result in the demonstration that Malta fever is by no means a rare disease in the warmer portions of the United States, and that many of the so-called anomalous cases of typhoid fever are in reality instances of infection with the organism of Malta fever.

Weil and Ménard reported a sporadic case of Malta fever. The patient, a Parisian, had spent a few weeks in a country district where Malta fever was unknown. He had drunk about a liter of cow's milk a day and often ate cheese made from cow's milk. The clinical symptoms led to a diagnosis of Malta fever, and the patient's serum reacted with melitensis in a dilution of 1:1,700. The infecting organism was not obtained. The authors state that they do not know whence the contagion could have come. They are led to think that the “ melitococcus” must be more common than it has been thought to be.

In his report of the finding of agglutinins for the Malta fever organism in cow's milk, Kennedy states:

I think the possibility of a melitensis infection of cows in this country should not be lightly thrust aside. I have heard of two cases of undulant fever in people who have never been out of England, and it is possible there are others undiagnosed.

Khaled states that he has seen cases of undulant fever in Egypt in persons who never had a chance to ingest goat's milk, and yet they suffered from typical Malta fever as confirmed by laboratory diagnosis.

Klimmer and Haupt report that observations have been made in different places that perfectly healthy wives of farmers had miscarried without any evident cause. Later inquiries revealed that the cattle were infected with contagious abortion and that the women had been drinking the raw cow's milk.

According to Rebagliati, Malta fever exists in certain localities in Peru where infected goats can not be concerned in the etiology. He regards flies as possible carriers, but does not consider the possibility that cow's milk may be responsible for the disease. No statements are made as to whether the Peruvian patients had been drinking cow's milk.

PREVALENCE OF BR. MELITENSIS IN COW'S MILK

In 1911 Schroeder and Cotton reported that they had found the infectious abortion organism in 8 of 77 samples of market milk tested (over 10 per cent) and in the milk distributed by 6 of 31 dairies (over 19 per cent). Schroeder afterwards reported a higher percentage of milk samples infected with Bacillus abortus. He injected into guinea pigs 516 samples of milk from 90 dairies, and 103 of the animals developed the abortion disease. The results showed that the milk from 29 of the dairies was infected from time to time with the abortion organism.

The findings of Schroeder and Cotton have been confirmed by other investigators in other sections of the United States. Fabyan, working in Massachusetts, examined the milk from 12 cows of a thoroughbred Guernsey herd. He found the abortion organism in two of the samples. Huddleson has noted its prevalence in cow's milk in Michigan, and Fleischner and Meyer have noted its prevalence in California. There is no doubt, therefore, that a large percentage of our population at some time or other have ingested living Br. melitensis of the bovine type.

That this organism is also present in cow's milk in Europe is evidenced by the findings of Zwick and Krage, Kennedy, and Winkler. Zwick and Krage, working in Germany, cultivated the contagious abortion organism on agar directly from the milk of three cows. Their report does not indicate how large a percentage of samples of milk contained this organism. Winkler also investigated the milk of cows in Germany. By inoculation of guinea pigs he demonstrated the specific organism in the milk from 13, or 41 per cent, of 32 cows which had recently aborted. He was able to obtain cultures directly from 3 of the samples of milk. He also demonstrated B. abortus in 7, or 32 per cent, of 22 samples of market milk.

In 1914 Kennedy, working in England, was testing goat's milk for agglutinins specific to Micrococcus melitensis, and found, to his

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