which ran a course parallel to that of the disease in man. The only logical conclusion which could be formulated from this work was that the Maltese goats were carriers of the virus of Malta fever and one of the principal means of transmitting the disease to human beings through the ingestion of their milk.

All the available evidence points to contaminated food as the vehicle by which these goats become infected with the virus of Malta fever. It has been definitely established that monkeys and goats may be infected in this manner. Furthermore, it has been shown that the urine of infected goats and of ambulatory cases in man at times contains the Micrococcus melitensis, so that goats feeding on material that has come in contact with such urine (which is not at all infrequent by the usual method of handling these animals) are readily infected. Thus the frequency and method of infection in goats are quite readily explained.

Other proofs of the transmission of Malta fever to man through the milk of infected goats, in addition to the confirmatory evidence furnished in this article, is forthcoming from a number of experiences which have been noted since regulations were enforced looking to the elimination of goats' milk as a source of infection. Thus the disease is said to have practically disappeared from Gibraltar following the removal of the infected goats; the affection does not occur among the convicts of the civil prison at Malta who are not allowed milk, but Malta fever is nevertheless present in the district where the prison is located; all fresh milk now being supplied to the soldiers at Malta is pasteurized, with the result that Malta fever among them has been reduced 90 per cent, while the disease has not abated among the civil population which continues to use infected milk.

EXPERIMENTS WITH MALTESE GOATS' BLOOD.

The method employed at the commencement of this work in studying the agglutination property of Maltese goats' blood on the Micrococcus melitensis was the macroscopic sedimentation test, but this was found to be so unsatisfactory that after a few trials the microscopic agglutination test was substituted and used throughout the remainder of the tests.

Two different strains of Micrococcus melitensis were employed in this investigation, one procured from the United States Public Health and Marine-Hospital Service and the other from the laboratory of the United States Army Medical Museum. In the early tests the dilutions used by the Mediterranean-Fever Commission in its routine work—which were as low as 1-10 and 1-20-were employed, with the result that a very high percentage of the animals tested reacted. This result led to the conclusion that the dilution may not have been sufficiently great, and we proceeded to try the same dilu

tions with the blood of an American goat known to be free from infection with Micrococcus melitensis as a check on the agglutination work.

EFFECT OF NORMAL AMERICAN GOATS' BLOOD ON MICROCOCCUS MELITENSIS.

A specimen of blood was obtained under sterile conditions from American goat No. 15, kept at the Bureau of Animal Industry Experiment Station at Bethesda, Md. To 15, 20, and 25 c. c., respectively, of sterile physiological salt solution 1 c. c. of this serum was added, making dilutions of 1-15, 1–20, and 1–25. One platinum loopful of each of these dilutions was then added separately to one loopful of a bouillon culture of Micrococcus melitensis 48 hours old and thoroughly mixed on cover glasses. From these the three hanging-drop preparations were made, thus making the final dilutions 1-30, 1-40, and 1-50, respectively. They were watched carefully for signs of agglutination. A control consisting of a hanging drop of the bouillon culture without any added serum was placed under a 4 mm. lens. At the beginning of the experiment the organisms in all four preparations were free and scattered homogeneously in the fluid throughout the microscopic field.

In the 1-30 dilution agglutination was well marked in 1 hour, and in 2 hours it was perfect, with large clumps and no free organisms. In the 1-40 dilution agglutination was slight after 1 hour, and in 2 hours small clumps were numerous, with some free organisms.

In the 1-50 dilution after 1 hour no agglutination was present, and in 21 hours a few very minute clumps could be seen, although the great majority of the organisms were free. In 3 hours the clumps were more numerous, but even this late it would never be mistaken for a reaction.

The control remained perfectly homogeneous for 3 hours, when it was discarded.

The above test showed that even normal goats' serum agglutinated Micrococcus melitensis to a certain extent. Therefore in examining the Maltese goats' blood, dilutions at least as great as 1-50 were considered essential. It also seemed important to place a time limit on the reaction. From other preliminary trial tests it was decided to use a dilution of 1-70 with a time limit of 11⁄2 hours and only consider animals to be infected when they would react under these conditions.

FEEDING EXPERIMENTS WITH NORMAL AMERICAN GOAT.

An experiment was undertaken to ascertain if it were possible to infect a normal American goat by means of the ingestion of Micrococcus melitensis in its food.

On October 14, 1905, American goat No. 4, whose blood had been tested and found not to react with dilutions as low as 1-40, was fed

at the Bureau Experiment Station with the contents of an agar plate containing numerous colonies of Micrococcus melitensis, together with one bouillon culture of this organism. Six days later three more bouillon cultures were fed, and on October 22 the final feeding of three additional cultures was made. Twenty-four days after the first feeding a small quantity of the blood was taken from the jugular vein and tested with Micrococcus melitensis agglutination. The first dilution tested was 1-60, and a well-marked clumping was produced. Dilutions 1-100, 1-150, 1-200, 1-300, 1-400, 1-500, 1-600, and 1-700 were then tried. Up to the high dilution of 1-400 this goat's blood, which had not reacted before the feeding with a dilution of 1-40, now gave a well-marked reaction. In all of the three higher dilutions clumping occurred, but these cases could only be classed as imperfect reactions, since the clumps were small, and many free organisms were present.

The result of this experiment, together with the similar findings of the Mediterranean-Fever Commission, left no doubt as to the danger of infection of goats from ingestion of Micrococcus melitensis.

AGGLUTINATION EXPERIMENTS.

AGGLUTININS IN SERUM OF RABBIT INOCULATED WITH MICROCOCCUS

MELITENSIS.

In order to have a constant supply of serum at hand for experimental purposes and for testing any suspected culture of Micrococcus melitensis, rabbit No. 1657 was inoculated intravenously in October, 1905, with 0.2 c. c. of a bouillon culture of this organism. In ten days its serum was tested and found to produce a well-marked agglutination of Micrococcus melitensis organisms in dilutions as high as 1-100. The rabbit, however, remained healthy and was finally chloroformed over fourteen months later. Culture media were inoculated with the blood and spleen at that time, but they remained sterile and were discarded after seventeen days' incubation.

EXAMINATION OF BLOOD OF GOATS AT ATHENIA QUARANTINE STATION.

With the knowledge acquired from the previously described check experiments, further examinations were made of the blood of the Maltese goats, using the higher dilution and time limit already specified. Specimens of blood were drawn with aseptic precautions by tapping the jugular vein with a small trocar and canula and were then placed in sterile glass vials. The following morning the clear serum had separated in all the containers, and it was then tested for its agglutination properties. These examinations were made of all the adult animals at regular intervals of two weeks, those reacting being placed in hospital quarters removed from the nonreacting animals.

The terms "good," "imperfect," and "no reaction" were used to designate the results of the test. A good reaction was considered to be one in which inside of the time limit of one and one-half hours the serum at a dilution of 1-70 produced a clumping of the bacteria in large clumps with no free organisms remaining in the field. Imperfect reaction was applied to those in which at the end of one and one-half hours small clumps were present but, in addition, some free organisms remained. No reaction was one in which the micrococci remained homogeneous in the hanging drop with no evidence of clumping.

It is probable that all of the imperfect reactors either were or had been infected with Micrococcus melitensis, as, upon making repeated examinations at varying intervals of time with blood from the same goat, it was occasionally found that a good reaction would be obtained while at other times it would be imperfect, and vice versa. By the above method of classification the 44 goats remaining at Athenia were examined up to December 22, 1905, with the result that 11 gave a good reaction, 9 an imperfect reaction, and 24 no reaction. The other 16 goats of the shipment not included in this number (5 having died on the ocean voyage to this country) will be referred to below.

EXAMINATION OF GOATS AT COLLEGE PARK.

Early in November, 1905, the herd of goats at Athenia was divided, 16 of the nonreacting animals being shipped from Athenia to the Maryland Agricultural Experiment Station at College Park and kept there under quarantine. Samples of blood were drawn from these 16 animals at different times under the same conditions as have already been described, with the result that by December 2, 1905, 6 gave a good reaction, 5 an imperfect reaction, and 5 failed to react. These 6 good reactors at College Park were then destroyed and the remaining 10 animals again tested two weeks later for the blood reaction. At this test goat No. 88, which on the previous examination gave an imperfect reaction, now gave a good reaction, and Nos. 81 and 91, which before gave no reaction, now gave an imperfect reaction. The results consequently were: Good reaction, 1; imperfect reaction, 6; no reaction, 3.

The infection therefore seemed to be spreading despite the fact that isolation and disinfection were practiced, and only 3 instead of 5 could be positively pronounced free from the organisms of Malta fever. This was not surprising, as the cold weather lowered the vitality of the animals, and they had previously been constantly subjected to infection from the ingestion of forage soiled by the urine of the reacting goats. These College Park animals were subsequently moved to the Bureau Experiment Station at Bethesda, Md.

EXAMINATION OF MILK OF REACTING GOATS.

From the fact that such a large proportion of reactions resulted from the agglutination tests, it was at once decided to endeavor to isolate the specific organism from the milk of these goats and thus demonstrate more forcibly the danger of their spreading Malta fever in the United States.

On December 11, 1905, milk was obtained in sterile wide-mouthed glass bottles from goats Nos. 19, 40, 54. 58, 65, 82, 84, and 85, all of which had given either a good or an imperfect agglutination reaction. The samples of milk were brought to the laboratory and plated at once in the ordinary manner of isolating organisms from mixed cultures, three plates being used for each specimen of milk. After four days' incubation many suspicious colonies were found on the plates from goats 19 and 40. Cultures were made from these colonies and the organisms put through the ordinary determinative tests, for classifying bacteria. In both cases the organisms were found to be decolorized by Gram's stain and grew on gelatin without liquefaction and in litmus milk without altering the medium. The final proof that they were the Micrococcus melitensis was secured when on testing them with specific serum from an infected animal they were agglutinated in large clumps. The plates made from the other animals were negative for Micrococcus melitensis.

This experiment proved beyond doubt that at least 2 of the 8 goats examined were excreting Micrococcus melitensis in large numbers in their milk, which was perfectly normal to all appearances and chemical analysis. Subsequent examinations of other reacting goats revealed the presence of this micrococcus in the milk of 2 additional animals, making a total of 4 goats that were eliminating the Micrococcus melitensis in their milk.

EXAMINATION OF URINE OF REACTING GOATS.

From a number of the reacting and imperfectly reacting Maltese goats, urine was collected in as aseptic a condition as possible in the following manner: After washing off the external genitalia with a 5 per cent carbolic-acid solution, a sterile catheter was passed along the floor of the vagina to the urethra and into the bladder. The urine was collected in sterile glass bottles and taken to the laboratory and plated at once in the same manner as had been done with the milk. Of the 11 specimens examined, Micrococcus melitensis was recovered from 1-goat No. 40-which animal was also known to be excreting the organisms very plentifully in its milk.

The fact that only one of the animals examined showed the presence of the organism in its urine is not surprising when we consider the result of similar examinations by the Mediterranean-Fever Com