Vol.

40

-The

Brucella

Ring

Test*

Its Potential Value in the Control of Brucellosis ADDIE V. HAMILTON, M.S., AND ALBERT V. HARDY, M.D., F.A.P.H.A.
Bureau of Laboratories, Florida State Board of Health, Jacksonville, Fla., and Department of Bacteriology, University of Florida, Gainesville, Fla.

U NDULANT fever remains with us are easy to collect. Now there is at hand as an increasing rather than a de- a simple and more reliable test which creasing problem. Its incidence and may be performed on specimens easy to epidemiology vary widely by area and obtain. occupation, but for the country as a The Brucella Ring Test was described whole, the commonest source of infection in 1937 by Fleischhauer. Only since the is the dairy cow and the commonest termination of hostilities has it been mode of transmission is the ingestion of studied widely. In this country, evaluamilk containing viable Brucellae. Thus, tion trials began in 1948. The test is the identification of milk from infected based on the fact that in whole milk the cows is a matter of substantial public agglutinated organisms adhere to the fat health importance. The Brucella Ring globules and are carried to the surface Test provides a new testing procedure. as the cream rises. A stained antigen is It is ingenious though simple. We be- employed. In a positive test, the cream lieve its practical value in control pro- ring is colored by the agglutinated grams warrants careful evaluation. As stained organisms. The milk below is yet, we have no fixed conclusions as to white, or at most, slightly tinted. In. the its proper place or relative value. Our negative test, by contrast, the cream observations are presented briefly, as a ring is uncolored while the milk is progress report. heavily colored. The test is simple to Serum agglutination is the standard perform. One ml. of milk is placed in procedure for detecting Brucella re- a test tube and one drop of stained actors. For the collection of blood speci- antigen is added. This is mixed by a mens, the time of the veterinarian and brief shaking. The test is incubated at those assisting him is required; for the 370 C. for 30 to 60 min. and read. performance of the tests, qualified techAntigen preparation is the initial and nical workers are needed. There is a most important step in technique. Foltemporary but significant reduction in lowing comparative studies, we have used milk flow following bleeding. This test Brucella abortusi, strain 19, Bureau of is not inexpensive. Whey agglutination Animal Industry, for all antigen producis more difficult to perform and findings tion. The organisms were grown on are of less significance. Its one advan- potato infusion agar for 48 hrs., were tage is that the samples to be examined washed off with 0.5 per cent phenolized saline, then washed and collected by *Presented before the Laboratory Section of the centrifugation. The bacteria were stained American Public Health Association at the Seventy- in a 1:5 dilution of Delafield's hemaseventh Annual Meeting in New York, N. Y., October toxylin for 5 min. Best results were 25, 1949.
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AMERICAN JOURNAL OF PUBLIC HEALTH

Mar., 1950

obtained on mixing 10 ml. of packed bacterial cells in 1,200 ml. of stain. The organisms were collected by centrifugation and the excess stain removed by repeated washing in acidified tap water. The final antigen is a suspension of stained Brucella abortus, 4 per cent by volume, in a 50 per cent glycerin-50 per cent phenolized saline mixture. Variation in the sensitivity of different lots of antigen prepared from the same organism have been noted. A standardized antigen, prepared and distributed by an official agency, would aid greatly in the evaluation of this test. Ordinarily, the ring test is performed on undiluted whole milk. Agglutination titers may be obtained by diluting the sample in series using known negative whole milk as the diluent. We feel that this quantitative procedure is needed in accumulating comparative data. The ring test has been recommended chiefly for use in testing mixed milk samples. Data on individual animals, we believe, will be more informative. Findings by the blood test, the whey test, and the ring test have been studied comparatively. Our present impressions are these: Cows strongly positive by blood test, are almost invariably also positive by ring test; conversely, those strongly positive by ring test are almost always positive by blood test. Any discrepancy in findings, with occasional exceptions, is in those animals which have weakly positive or doubtful reactions by one or the other test. The blood titer on an individual animal is ordinarily above the ring test titer, and commonly much higher. There is however, no regularity in the relationship. The two may show closely similar titers; on the other hand, the blood titer may be high while the ring test is positive only in undiluted milk. Since a positive ring test on an individual cow is often obtained only on undiluted milk, the limitations in value of a test on mixed milk samples is evident.

We have found the whey agglutination to be less sensitive than the ring test. Specimens with weak positive reactions by ring test were often negative by whey

agglutination. The ring test proved surprisingly reliable in identifying infected herds on examining a mixed sample from the herd. Presumably this is due in part to the fact that in this communicable disease, a considerable percentage of the animals in an infected herd are infected. In 67 of 68 herds studied, there was agreement between blood findings and ring tests performed on mixed milk samples. In one case, a weakly positive ring test was obtained on a herd presumed to be free of Bang's infection. Calfhood vaccination had been practised, but ordinarily such animals have few or no agglutinins in serum or milk. This herd was not re-bled to confirm the negative findings by serum agglutination. Conditions which might effect ring test readings have been studied. Results on the raw and the subsequently pasteurized milk were essentially identical. Shipped specimens with added preservative were satisfactory for testing. Tests on animals with mastitis were difficult to read and in obviously abnormal milk gave what appeared to be a false-positive reaction. In animals, vaccinated as adults, agglutinins were present which made it difficult to interpret findings. Our studies to date lead us to conclude, that ring tests on milk collected from individual cows, or on mixed samples from groups of cows, provide findings in close agreement with those obtained by serum agglutination tests. We feel, therefore, that it has practical value. The precise role of this test in a brucellosis control program will be defined only by extensive experience. Present statements can have the weight only of individual opinion and suggestions. Raw milk is still used-and will continue to be consumed by rural families

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and in villages. Certainly every sample of milk from a supply for raw consumption which comes to a public health laboratory should be examined by a ring test. We believe there should be a concerted effort to have the milk of all "family cows" on our farms and in villages tested by ring test if a blood test has not been performed. Using preservative, specimens can be collected in one ounce bottles-such as are used for fecal specimens-and mailed to the laboratory. The ring test is essentially an examination for the probable presence of a pathogenic organism in milk. If the early promise of this test is confirmed, it will be more nearly practicable to protect our public from the use of raw milk from Brucella infected cows. The second major value of the ring test will be in providing easy and frequent checks of clean herds. This will facilitate an early recognition of introduced infection. Thus, the problem of reestablishing control would be held at a minimum. In the absence of a general blood testing program, the ring test will differentiate presumably clean from presumably infected herds. Limited facilities and resources for blood testing could be

used where of greatest probable value. To what extent the ring test may be substituted for blood tests on individual cows can be determined only by extensive comparative studies. If the tests prove of nearly equal value, the one which uses specimens which are easy to collect will be utilized more readily and probably more frequently. The ring test we view as an effective screening procedure. The screen will be fine if samples come from individual cows, it will be very coarse if one sample of the mixed milk of a whole herd is tested, it will be a moderately effective screen if a sample from each can is collected. The ring test as a screen test will not replace the blood test; but it will minimize unproductive blood testing and will increase the importance of every blood test which is performed. We recommend the ring test for trial and study, with emphasis on the latter. No test procedure available is wholly reliable. Certainly we must have secure knowledge as to the comparative value of the ring test and blood tests in indicating udder infection and the shedding of Brucella in milk. We are confident the ring test has merit; it may have high practical value.

Royal Sanitary Institute

The 1950 Annual Congress of the Royal Sanitary Institute will be held at Eastbourne, April 24-28. Fellows and members of the American Public Health Association who expect
to be in England during the time of this meeting are asked to communicate with the Executive Secretary, American Public Health Association, well in advance of that time.