> THE LABORATORY IN THE DIAGNOSIS OF BRUCELLOSIS*

Transcription

1 > THE LABORATORY IN THE DIAGNOSIS OF BRUCELLOSIS* ". WESLEY W-. SPINK, M.D. From the Department of Medicine, University of Minnesota Hospitals and Medical School, Minneavolis. Minnesota A precise diagnosis of human brucellosis can only be made with the aid of laboratory procedures. There are no distinguishing clinical characteristics of either acute or chronic brucellosis that will permit even the most astute clinician to make more than a presumptive diagnosis of active disease without help from the laboratory. Because the signs and symptoms of brucellosis are not specific, a great responsibility is placed upon laboratory personnel by the clinician attempting to resolve a diagnosis of brucellosis, and the clinician in turn must interpret with due caution any laboratory report. Before attempting to evaluate the laboratory tests for brucellosis, it is well to review briefly the features of brucellosis that first prompt the physician to entertain the diagnosis, and then to summarize the present status of diagnostic procedures that establish either the presence or absence of active disease. This summary is based largely upon experience in the diagnosis and management of patients at the University of Minnesota Hospitals over a period of fifteen years. During this time clinicians at the University Hospitals have been very fortunate in having the cooperation and aid of two groups on the University campus, who have been interested in diagnostic procedures in both animal and human brucellosis. These include members of the Division of A r eterinary Science and the personnel of the Laboratories of the Minnesota Department of Health. THE PRESUMPTIVE DIAGNOSIS OF BRUCELLOSIS A diagnosis of brucellosis is considered by the clinician on the basis of certain clinical and epidemiologic features. One of these is fever of undetermined etiology. The acutely ill patient with fever may have only a mild infection but usually complains of weakness, sweats, chilly sensations and vague aches and pains. The more seriously ill patient will have these symptoms in accentuated form, with localizing manifestations of brucellosis such as cervical and axillary adenopathy, splenomegaly, and pain over the spine, especially in the lumbar area. Obviously many infectious diseases provoke the foregoing symptoms. The differential diagnosis is commonly quite difficult because the patients do not exhibit any abnormal physical findings. The more chronically ill and febrile patient with brucellosis presents weakness as his primary complaint, along with sweats, headache, backache, nervousness, insomnia and mental depression. Most of these patients also present a perplexing diagnostic problem to the clinician because there are no characteristic physical findings. Any number of diseases can provoke such a picture including tuberculosis, lymphoblastoma and carcinoma. * Prepared for presentation at the Thirtieth Annual Meeting of the American Society of Clinical Pathologists, in Chicago, October 17, rieceivod for publication, November 23,

2 202 SPINK Those physicians living in endemic areas of animal brucellosis, and especially those interested in the disease, may suspect the presence of brucellosis in patients presenting the foregoing complaints, and will immediately call upon the laboratory for confirmation. But because brucellosis most often occurs sporadically in the United States, and because most physicians seldom encounter proved cases of brucellosis in their every day practice, the diagnosis is frequently overlooked or delayed until the more common causes of fever are explored. However, if the following epidemiologic data relating to brucellosis are borne in mind by the clinician when he encounters a patient with fever of doubtful etiology, the diagnosis may be suspected. Brucellosis is primarily not a human disease. The reservoir is in farm animals, and in the United States, chiefly cattle and swine. Human beings contract the disease through either direct or indirect contact with infected animals. Studies carried out in Minnesota, 28 Wisconsin 45 and Iowa 2 have I revealed that approximately three-fourths of all human infections are due to I direct contact with either infected animals or their immediate environment. Not more than one-fourth of instances of human brucellosis originates from the ini gestion of contaminated milk or milk products. Approximately three-fourths of j the infections occur in males. Although children may be frequently exposed to the disease, brucellosis is not recognized as a common infection in the younger age groups. The clinician should suspect the presence of the disease in those persons whose occupation brings them into contact with infected animals, including farmers, rural housewives and employees of meat-packing plants. Much less frequent is active disease in adult persons who have become infected through drinking milk. DIAGNOSTIC LABORATORY PROCEDURES The usual initial procedures in attempting to resolve a correct diagnosis in any febrile patient are to analyze a fresh specimen of urine, and to examine the peripheral blood. The results of a routine urine examination are of no aid in the diagnosis of brucellosis. This also applies to the hemoglobin determination, and to the erythrocyte count. However, the total and differential leukocyte counts may reveal quite valuable information, and may present the first clue to the possibility of brucellosis being present. In brucellosis the total number of leukocytes is usually normal or reduced, being infrequently elevated above 10,000 cells per cu. mm. The differential count very often reveals a relative increase in the lymphocytes to 40 to 60 per cent of the total white count. A few of the lymphocytes may present atypical forms such as are found in infectious mononucleosis. 38 r The sedimentation rate of erythrocytes is of no diagnostic value in brucellosis. 1 Normal, as well as accelerated, rates may be present. If elevated, the sedimentation rate may be of some help prognostically, particularly in relation to the effects " of antibiotic therapy. The Agglutination Test I Since the agglutination test for brucellosis was first introduced by Wright, 44 1 this reaction has proved to be the most valuable laboratory procedure for rapid

3 DIAGNOSIS OF BRUCELLOSIS 203 screening. At the University Hospitals two agglutination technics are employed. The first is a rapid-slide agglutination test, utilizing an antigen prepared by Dr. M. Ruiz Castaneda of Mexico City. This antigen consists of Brucella abortus cells fixed with formalin, and then suspended in saline solution to which has been added methylene blue. The test is performed by placing a drop of the patient's whole blood on a glass slide and mixing it with a drop of the antigen. Within 30 seconds, a peripheral blue-green ring signifies the presence of brucella agglutinins. The antigen has been titrated by the tube-dilution agglutination method so that in performing the slide test clumping of cells takes place only in the presence of a serum diluted 100 times or more. In other words, bloods having agglutinins present in a titer below 1:100 by the tube-dilution technic will not show a positive slide test. This rapid-slide test has been correlated with the tube-dilution test in several hundred persons, and there has been remarkable agreement of results. The second agglutination technic is the tube-dilution test, employing an antigen of Br. abortus supplied by the Bureau of Animal Industry of the United States Department of Agriculture. No matter what results are obtained with the slide test, the tube-dilution agglutination test is performed with every blood from persons suspected of having brucellosis. After optimal dilution of the antigen and serum, the tubes are placed in a water bath at 37 C. for 48 hours. The end point is the tube containing the highest dilution of the serum with complete clearing of the suspended antigen and sedimentation and clumping of the cells. In recent years, the efficiency of the agglutination test for diagnostic purposes has been questioned. The origin for some of this skepticism stems from the widespread application of intradermal diagnostic tests in the diagnosis of doubtful cases of chronic brucellosis. Many of these persons giving a positive skin' test either do not possess demonstrable agglutinins, or agglutinins only in a low titer. The tendency then is to discredit the agglutination test as a diagnostic tool, rather than to question the reliability of the intradermal reaction. We have seen a large number of patients in our clinics in whom the diagnosis of chronic brucellosis has been made elsewhere upon the basis of nonspecific symptoms and a positive intradermal test with brucella antigen. In these patients, agglutinins are usually absent, or present in low titer. After repeated contacts with such patients, one cannot escape the conviction that too many physicians are over enthusiastic and uncritical in the application and interpretation of the skin test. Since this procedure is not really a laboratory test, it will not be discussed further, except to state our viewpoint concerning its significance and value as a diagnostic aid. The evidence at hand indicates that a positive intradermal reaction to brucella antigen is a manifestation of previous invasion of the tissues by Brucella. Regardless of the symptoms, a positive test does not mean the presence of an active infection. Furthermore, one is not justified in making a diagnosis of brucellosis if a dermal reaction is followed by an elevated titer of brucella agglutinins. Because from 10 to 20 per cent of the normal population in an endemic area of brucellosis will reveal a positive reaction, we have abandoned the skin test as a diagnostic procedure. It does have a useful place as an epidemiologic tool for determining the incidence of exposure of a given population to the disease.

4 ;204 SPINK.' Another reason that has been given for doubting the dependability of the agglutination test as a diagnostic procedure is that patients with no demonstrable agglutinins may exhibit positive blood cultures for Brucella. And yet, if one attempts to seek the documented evidence for this statement, there is little to support it. In our own experience, only on' very rare, occasions has Brucella been isolated from the blood stream when agglutinins were absent. Investigators in this country who have had extensive experience with diagnostic methods for brucellosis, such as McCullough 26 and Jordan and Borts, 2 ' 24 have come to the ' same conclusion. Attention should be called to the dissenting viewpoint of West and Borman, 43 of the Laboratories of the Connecticut State Department of Health, who found no agglutinins in 16 per cent of persons who had Brucella in specimens of their clotted blood. No details of the agglutination test were given in the report. This finding stimulated Damon and Albright 8 of the Laboratories of the Indiana State Board of Health to carry out a similar study. These workers came to just the opposite conclusions after examining several thousand specimens of blood. They stated, "It is evident that culturing of all agglutination test clot specimens for Brucella is unprofitable as only two recoveries were obtained from specimens which failed to show agglutinins in a dilution of 1:40 of the patients' serum." A question that is frequently asked is, "What is a significant diagnostic titer of agglutinins?" Though one cannot present an unqualified answer, it has been our experience that the higher the titer of agglutinins, the more likely it is that \ one is dealing with an active case of brucellosis, and positive blood cultures are! much more commonly associated with blood having a high titer of agglutinins. ' In an analysis of a large number of cases in Minnesota, it was observed that over 90 per cent of the patients having positive blood cultures had simultaneous agglutinin titers of 1:320 or above. 28 A final answer to this question is dependent largely upon the sensitivity of the antigen that is employed, and the technic that is applied in performing the test. In our laboratory, it is uncommon for us to establish the diagnosis of brucellosis when blood cultures remain sterile, and when the agglutinin titer with several specimens of blood from a suspected case remains at 1:80 or below. This immediately leads to another question, "What is the significance of a low titer of agglutinins, particularly in patients with illdefined symptoms?" It has been found that in an endemic area of brucellosis a considerable proportion of normal persons possess a low titer of brucella agglutinins. Spink and Anderson 37 demonstrated that 18.5 per cent of 1627 donors of blood coming to the University of Minnesota Hospitals had agglutinins for Brucella, but less than 2 per cent had titers above 1:80. It is of interest that most of these donors came from rural areas, and roughly three-fourths of them were males. The presence of a low titer of agglutinins in such a large number of presumably normal persons could be interpreted in one of several ways. First, these subjects may have been exposed to brucellosis in the past, and infection with recovery had occurred. In a recent analysis of over 100 persons with bacterio- Jogically proved brucellosis in our clinic, it has been demonstrated that a low titer of agglutinins will persist in the blood for many months, and even years, after apparent recovery. Second, these low titers may have been stimulated by \

5 DIAGNOSIS OF BRUCELLOSIS 205 repeated contact with brucella antigen in various ways, including the ingestion ' of milk containing killed Brucella. In fact, in this clinic Braude, Gold and Ander- \ son 3 caused brucella agglutinins to appear in the blood of persons by having them ^ ingest milk to which heat-killed brucella cells were added. Such a procedure stimulated the production of agglutinins but it did not result in dermal hypersensitivity. However, others have contended that the ingestion of heat-killed brucellaein milk does not result in significant titer of agglutinins. 27 Third, these low titers of agglutinins for brucella may represent nonspecific reactions to Brucella. It is well established that the brucella agglutination test is not a specific reaction. Cross reactions with anti-brucella serum occur with Vibrio cholerae ' and with Bacterium tularense. It has been shown that brucella agglutinins will»u appear in persons immunized with cholera vaccine. 10 Occasionally, patients with 1 ' tularemia will reveal the presence of brucella agglutinins in their blood, and patients having brucellosis may have agglutinins for V. cholerae. Further evidence that the presence of brucella agglutinins may at times represent a nonspecific reaction in cattle has been reported by Hess and Roepke, 20 who utilized filter paper chromatography of bovine serum, as suggested by Castaneda. 6 They were ' able to demonstrate a nonspecific agglutinating material for Brucella, particularly in those serums having a low titer of agglutinins. Our own interpretation of a low titer of brucella agglutinins, at least in the majority of human serum, is that it represents past exposure to brucellosis. A discussion of. the clinical significance of the agglutination reaction cannot be concluded without reference to the prozone phenomenon and to the problem of blocking antibodies for Brucella. Occasionally, a serum is encountered in which there is absence of agglutination in those tubes with the lower dilutions of serum. Thus, agglutinins may be absent in dilutions of from 1:20 to 1:160. For this reason, we recommend that every tube-dilution test should be carried out with dilutions up to at least 1:640 or 1:1280. Griffitts 17 first called attention to the presence of a blocking mechanism for brucella agglutinins in human serum. These observations have been extended by Schuhardt, Woodfin and Knolle. 36 There appears to be little doubt that occasionally such a mechanism is present, but the blocking of agglutinins involves the lower titers, and it appears to be of little practical significance in the diagnosis of brucellosis. Since the agglutination test is such an important diagnostic procedure it is well to review some of the pertinent factors that influence the reaction. Emphasis is placed upon the tube-dilution technic since in our experience it has been difficult to obtain reliable quantitative data with the rapid-slide test, although the previously described Castaneda procedure is valuable for the preliminary investigation of suspected cases. Three principal factors are involved in the test, namely, the antigen, the serum, and the technic. The results of dependable and comprehensible investigations of these various factors are embodied in a series of papers by Fitch and associates, 12 ' 13 which should be consulted for detailed information on the subject. These studies were concerned with bovine serum but similar conclusions have been reached by others working with human serum. 0 ' " 33 As far as the antigen is concerned it is not necessary to employ homologous

6 206 SPINK brucella preparations for the agglutination test. Any properly prepared smooth strain of Brucella appears to be adequate. 11 It should be pointed out that antigens containing small amounts of agar will give false-positive tests. At the Brucellosis Research Center of Montpellier, France, where investigations have been carried out for many years, a strain of Brucella suis is employed for the antigen, although practically all the human disease is due to Brucella melitensis. In Mexico City, where all the human infections are likewise due to Br. melitensis, a strain of Br. abortus is utilized. 7 In our clinic, where we are dealing primarily with infections due to Br. abortus, we employ Br. abortus for an antigen, although similar results with the agglutination test have been obtained with the Montpellier antigen. There is no reason for employing viabler bucellae in the test. The organisms may be killed with formalin, phenol or heat, each procedure having certain advantages and disadvantages. It is important in carrying out the test to add optimal concentrations of the antigen to the serum. This factor probably accounts for some of the discrepancies in results of different laboratories. The properties of the serum influence the outcome of the agglutination test. Serum that is severely hemolyzed will yield false-positive reactions. If there must be some delay in performing the tests, serum should be removed from the clot and stored in clean sterile tubes at refrigerator temperature. The capacity of a serum to agglutinate will not deteriorate when kept in a frozen state. Important factors in determining the outcome of the agglutination reaction are related to time and temperature. Fleming 14 called attention to the fact that the union of agglutinin and antigen is hastened by elevations of temperature between 18 and 55 C. This observation has been confirmed many times. To obtain optimal results, one of three methods of incubation in a water bath may be carried out; first, incubation at 37 C. for 48 hours; second, incubation at 55 C. for 24 hours; third, incubation at 37 C. for 24 hours and then centrifuging the antigen-serum mixtures. It is surprising how lack of experience in reading the agglutination tests causes discrepancies in the results. Cultural Methods for Brucella The most reliable laboratory procedure in the diagnosis of brucellosis is isolation of Brucella from the patient. Many clinicians and laboratory workers frequently express disappointment over their inability to recover Brucella from patients with active brucellosis. This presumed failure may be related to several causes. Of paramount importance is the fact that at the time when blood was obtained for culture no bacteria may have been present in the circulating blood. If at all possible, multiple blood specimens from a person with suspected brucellosis should be obtained for culture. However, it should be remembered that in the human disease, only a relatively few organisms are present in the blood at any one time. This feature was recognized by members of the Mediterranean Fever Commission. 19 As a result of the small numbers of brucellae which they found in blood cultures, they abandoned the theory that the disease was transmitted by blood-sucking insects. Another major factor in the success of culturing

7 DIAGNOSIS OF BRUCELLOSIS 207 Brucella is employment of a proper growth medium. Various cultural media have been proposed, and many have been investigated quantitatively in our laboratory. At the present time we are not using liver infusion and tryptose media because it has been shown that certain batches of each may inhibit the growth of Brucella. 4 ' 32 ' 34 Furthermore, we have obtained more favorable growth with Trypticase soy broth* and with "Albimi" medium.f The latter appears to be particularly favorable for the growth of Br. melitensis. For culturing Brucella from blood or other body fluids we employ the method of Castaneda. 5 Approximately 15 to 20 ml. of broth is introduced into rectangular, rubber-stopped bottles having a capacity of.120 ml. Agar in a concentration of 3 per cent is first dispersed along one of the inner sides of the bottle and allowed to harden before introducing the broth. To each container is added 5 ml. of blood, which is mixed with the broth, and then the mixture is distributed over the agar for a moment or two. Carbon dioxide is introduced into the bottle with the aid of a needle in a concentration of 10 per cent. The bottles are then set upright in the incubator at 37 C. and examined daily. If no colonies have appeared on the agar within 48 hours, the bottle is gently tipped and the contents are allowed to remain in contact with the agar for a brief period of time, the bottle being replaced in the incubator again in the upright position. If brucella organisms are present, usually within a week colonies are noted growing on the agar. Such a cultural method has several advantages. It eliminates opportunities for contamination which are induced by multiple subcultures, and it protects laboratory personnel. Although most investigators have experienced difficulty in isolating Brucella even from persons with undoubted acute brucellosis, Pickett and Nelson 31 have recently claimed the isolation of nonsmooth brucella variants from the blood of febrile patients, who presumably did not have brucellosis, and from the blood of 100 normal persons. It is impossible to assess the significance of these extraordinary findings at this time, since confirmatory reports are lacking. It is well known that Brucella localizes in those tissues with an abundance of reticulo-endothelial cells, including the bone marrow. 41 Brucella has been recovered in this clinic from aspirated sternal bone marrow when simultaneous cultures of venous blood have remained sterile. Janbon and Bertrand 22 have emphasized that enlarged lymph nodes in the cervical region constitute additional evidence of active brucellosis, and in a large number of infections they have readily isolated Br. melitensis from surgically removed specimens of these nodes. 23 We have been unsuccessful in growing Brucella from excised specimens of liver although bacteremia was demonstrated in these patients. 40 We have not attempted, and we do not recommend the aspiration of splenic tissue for cultural purposes. Weed and Dahlen 42 have pointed out the advantages of carefully culturing tissue removed for biopsy, and in this manner have demonstrated Brucella in persons with unsuspected brucellosis. It has been known, though not generally recognized, that Brucella may be * Baltimore Biological Laboratory, Baltimore, Maryland, t Albimi Laboratories, Brooklyn, New York.

8 208 SPINK excreted in the human urine for long periods of time. 25,36 Meyer 30 has pointed out that Brucella localizes in the epithelium of Bowman's capsule and the convoluted tubules. More recently, Greene and Albers 16 have commented upon the similarity between brucella infection of the urinary tract and tuberculosis. They cultured Brucella from the urine in two patients. Our own efforts to isolate Brucella from the urine of patients with known brucellosis have been disappointing. In one instance of subacute bacterial endocarditis, Br. abortus was cultured from the urine. Although Brucella may localize in the biliary tract and cause cholecystitis, we have not succeeded in culturing Brucella from the bile of patients with active brucellosis. 29 Brucellosis is an important cause of chronic meningitis, and we have succeeded in culturing Brucella from the cerebrospinal fluid in such cases, when the blood cultures remained sterile. 39 We have confirmed the observation of Goodpasture and Anderson 16 that brucellae grow readily in the viable chick embryo, and we have utilized this method on occasion for growing brucellae from body fluids, pai'ticularly from cerebrospinal fluid. 18 ' 39 Opsonocytophagic Test This immune reaction has been advocated by some as a diagnostic procedure when evaluated with the results of the skin test and agglutination test. 21 No doubt, as a patient recovers from brucellosis, there is an increased capacity of the polymorphonuclear neutrophil leukocytes to phagocytize viable, smooth brucella cells, but the results of this test have been too inconstant in our hands to be of any practical value for the diagnosis of brucellosis. Complement-Fixation Test Complement-fixing antibodies usually parallel the presence of agglutinins. Quantitative complement-fixation tests have been carried out in this laboratory simultaneously with agglutination tests. No practical differences have been encountered between the results of the two tests. Therefore, since the agglutination test can be performed with less difficulty, the complement-fixation test has been abandoned in our laboratory. SUMMARY 1. The clinician can only make a presumptive diagnosis of brucellosis. A positive intradermal test with brucella antigen only denotes previous exposure to Brucella and, regardless of the symptoms, does not establish a diagnosis of active disease. A precise diagnosis is dependent upon laboratory procedures. 2. Helpful aids in screening suspected instances of brucellosis include total and differential leukocyte counts of the peripheral blood. Usually the disease is accompanied by a normal or reduced leukocyte count and a relative lymphocytosis. The erythrocyte sedimentation rate is of no diagnostic help. 3. The agglutination test is a valuable procedure for the diagnosis of brucellosis. The rapid-slide and tube-dilution tests have been described and evaluated.

9 DIAGNOSIS OF BRUCELLOSIS 209 A "diagnostic titer" of brucella agglutinins depends upon several factors, including the sensitivity of the antigen and the technic employed. It is unusual to encounter a patient with demonstrable bacteremia and absence of brucella agglutinins. 4. The most dependable diagnostic procedure is the isolation of Brucella from the body fluids or tissues. Success in culturing Brucella depends upon selection of a suitable culture medium and upon repeated attempts to isolate the organisms. Brucella cannot be isolated from a significant number of patients with undoubted brucellosis. 5. The opsonocytophagic test and complement-fixation test are not recommended as routine laboratory procedures. REFERENCES 1. AGNEW, S., AND SPINK, W. W.: The erythrocyte sedimentation rate in brucellosis. Am. J. M. Sc, 217: , BORTS, I. H.: Some observations regarding the epidemiology, spread and diagnosis of brucellosis. J. Kansas Medical Soc., 46: , BRAUDE, A. I., GOLD, D., AND ANDERSON, D.: Formation of antibodies in human subjects after the ingestion of heat-killed Brucella abortus. J. Lab. and Clin. Med., 34: , CAHRERE, L., RENOUX, G., AND QUATREFAGES, H.: A propos de Faction de certaines peptones (tryptose) sur les Brucella. Ann. Inst. Pasteur, 80: , CASTANEDA, M. R.: A practical method for routine blood cultures in brucellosis. Proc. Soc. Exper. Biol, and Med., 64: CASTANEDA, M. R.: Surface fixation. A new method of detecting certain immunological reactions. Proc. Soc. Exper. Biol, and Med., 73: 46-49, CASTANEDA, M. R., TOVAR, R., AND VELEZ, R.: Studies on brucellosis in Mexico. Comparative study of various diagnostic tests and classification of the isolated bacteria. J. Infect. Dis., 70: , DAMON, S. R., AND ALBRIGHT, K.: The isolation of Brucella from blood clots. Symposium on Brucellosis, American Association for the Advancement of Science, Washington, D.C., , EISELE, C. W., MCCULLOUGH, N. B., AND BEAL, G. A.: Discrepancies in the agglutination test for brucellosis as performed with various antigens and as reported from different laboratories. J. Lab. and Clin. Med., 32: , EISELE, C. W., MCCULLOUGH, N. B., BEAL, G. A., AND BURROWS, W.: Development of Brucella agglutinins in humans following vaccination for cholera. Proc. Soc. Exper. Biol, and Med., 61: 89-91, FBINBERG, R. J., AND WRIGHT, G. G.: Factors influencing the agglutination titration in human brucellosis. J. Immunol., 67: , FITCH, C. P., DONHAM, C R., BISHOP, L. M.. AND BOYD, W. L.: Studies of the test tube agglutination test for the diagnosis of Bang's disease (contagious abortion). Technical Bulletin 73, St. Paul: University of Minnesota Agricultural Experiment Station, pp FITCH, C. P., DONHAM, C. R., AND BOYD, W. L.: Further studies of the test tube agglutination test for the diagnosis of Bang's disease (contagious abortion). Technical Bulletin 77, St. Paul: University of Minnesota Agricultural Experiment Station, pp FLEMING, A.: On the influence of temperature on the rate of agglutination of bacteria. Brit. J. Exper. Path., 9: , GOODPASTURE, E. W., AND ANDERSON, L.: The problem of infection as presented by bacterial invasion of the chorio-allantoic membrane of chick embryos. Am. J. Path., 13: , GREENE, L. F., AND ALBERS, D. D.: Brucellosis of the urinary tract. Proc. Staff Meet-' Mayo Clin., 25: , GniFFiTTS, J. J.: Agglutination and an agglutinin blocking property in serums from known cases of brucellosis. Pub. Health Reports, 62: , HALL, W. 11., AND SPINK. W. W.: Therapy of experimental brucella infection in the developing chick embryo. I. Infection and therapy via the allantoic sac. J. Immunol., 59: , 1948.

10 210 SPINK 19.'HERROCKS, W. H., AND KENNEDY, J. C..: Mosquitoes as a means of dissemination of Mediterranean Fever. In Sect, vii, Reports of the Commission for the Investigation of Mediterranean Fever. Part IV. London: Harrison and Sons, pp HESS, W. R., AND ROEPKE, M. H.: A nonspecific Brucella-agglutinating substance in bovine serum. Proc. Soc. Exper. Biol, and Med., 77: , HUDDLESON, I. F.: Brucellosis in Man and Animals. New York: The Commonwealth Fund, p JANBON, M., AND BERTRAND, L.: Les adenomegalies de la brucellose humaine. Leur valeur diagnostique et pronostique. La Presse Medicale, 74: , JANBON, M., BERTRAND, L., AND QUATREFAGES. H.: L'adenoculture. Methode rapide et fidele de diagnostic bacteriologique de la brucellose aigue. La Presse Medicale, 77: , JORDAN, C. F., AND BORTS, I. H.: Brucellosis and infection caused bv three species of Brucella. Am. J. Med., 2: , KENNEDY, J. C: On the recovery of Micrococcus melitensis from the urine of Mediterranean fever patients. In Sect, iv, Reports of the Commission for the Investigation of Mediterranean Fever, Part III. London: Harrison and Sons, pp MCCULLOUGH, N. B.: Laboratory tests in brucellosis. Symposium on Brucellosis, Washington, D. C: American Association for the Advancement of Science, , MCCULLOUGH, N. B.. EISELE, C. W., AND BEAL, G. A.: Oral administration of killed Brucella to man. Pub. Health.Reports, 64: , MAGOFFIN, R. L., KABLER, P., SPINK, W. W., AND FLEMING. D.: An epidemiologic study of brucellosis in Minnesota. Pub. Health Reports, 64: , METTIEB, S. R., AND KERR, W. J.: Hepatitis and cholecystitis in the course of Brucella infection. Arch. Int. Med., 54: , MEYER, K. F.: Observations on the pathogenesis of undulant fever. In Essays in Biology. Berkeley and Los Angeles: University of California Press, p PICKETT, M'. J., AND NELSON, E. L.: Observations on the problem of Brucella blood cultures. J. Bact., 61: , POLDING, J'. B.: Some peculiarities in the germination of Brucella. J. Comp. Path. and Exper. Therap., 56: 215, SCHUBERT, J. H., AND HERNDON, J. F.: A study of certain factors affecting the agglutination test for brucellosis. Pub. Health Reports, 65: , SCHUHARDT, V. T., RODE, L. J., FOSTER, J. W., AND OGLESBY, G.: An anti-brucella factor in peptones. J. Bact., 57: 1-8, SCHUHARDT, V. T., WOODFIN, H. W., AND KNOLLE, K. C.: A heat-labile Brucella-agglutinin-blocking factor in human sera. J. Bact., 61: , SHAW, E. A.: Further observations on goats, cats, rats and ambulatory peases in connection with Mediterranean fever. In Sect, iii, Reports of the Commission for the Investigation of Mediterranean Fever, Part V. London: Harrison and Sons, 1907, pp SPINK, W. W., AND ANDERSON, D.: Brucella studies on bank blood in a general hospital. J. Lab. and Clin. Med., 35: , SPINK, W. W., AND ANDERSON, D.: Studies relating to the differential diagnosis of brucellosis and infectious mononucleosis: Clinical, hematologic and serologic observations. Trans. Assn. Am. Physicians, 64: , SPINK, W. W., AND HALL, W. H.: Encephalomeningitis due to Brucella abortus. Trans. American Clinical and Climatological Association, 61: , SPINK, W. W., HOFFBAUER, F. W., WALKER, W. W., AND GREEN, R. A.: Histopathology of the liver in human brucellosis. J. Lab. and Clin. Med., 34: 40-58, SUNDBERG, R. D., AND SPINK, W. W.: The histopathology of lesions in the bone marrow of patients having active brucellosis. Blood, Supp. No. 1, 7-32, WEED, L. A., AND DAHLEN, D. C: Bacteriologic examination of tissue removed for biopsy. Am. J. Clin. Path., 20: , WEST, D. E., AND BORMAN, E. K.: The culturing of blood clots for Brucella organisms. J. Infect. Dis., 77: , WRIGHT, A. E.: Note on the technique of serum diagnoses of acute specific fevers. Lancet, 1: 139, ZINTEK, A. R.: Brucellosis in Wisconsin. Wisconsin M. J., 49: , 1950.