•>• T H E LABORATORY IN T H E 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 Areterinary Science and the personnel of the Laboratories of the Minnesota Department of Health.
T H E P R E S U M P T I V E DIAGNOSIS O F
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, 1951.
rieceivod for publication, November 23, 1951.
201
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 Wisconsin45 and Iowa2 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
1
Since the agglutination test for brucellosis was first introduced by Wright,44
this reaction has proved to be the most valuable laboratory procedure for rapid
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.
;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 McCullough26 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 Albright8 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 Anderson37 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 bacterioJogically 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
\
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- \
son3 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. Griffitts17 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 ' "• 3 3
As far as the antigen is concerned it is not necessary to employ homologous
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. Fleming14 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
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 Nelson31 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 Dahlen42 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 L a b o r a t o r y , Baltimore, Maryland,
t Albimi Laboratories, Brooklyn, New York.
208
SPINK
excreted in the human urine for long periods of time. 25,36 Meyer30 has pointed
out that Brucella localizes in the epithelium of Bowman's capsule and the convoluted tubules. More recently, Greene and Albers16 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 Anderson16 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.
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 S P I N K , W. W . : T h e erythrocyte sedimentation rate in brucellosis. Am.
J . M . S c , 217: 211-214, 1949.
2. BORTS, I . H . : Some observations regarding the epidemiology, spread and diagnosis of
brucellosis. J . Kansas Medical Soc., 46: 399-405, 1945.
3. BRAUDE, A. I., GOLD, D . , AND ANDERSON, D . : Formation of antibodies in human s u b jects after t h e ingestion of heat-killed Brucella a b o r t u s . J . L a b . and Clin. Med.,
34: 744-750, 1949.
4. C A H R E R E , L., R E N O U X , G., AND QUATREFAGES, H . : A propos de Faction de certaines
peptones (tryptose) sur les Brucella. Ann. I n s t . Pasteur, 80: 321-322, 1951.
5. CASTANEDA, M . R . : A practical method for routine blood cultures in brucellosis. Proc.
Soc. Exper. Biol, and Med., 64: 114-115. 1947.
6. CASTANEDA, M . R.: Surface fixation. A new method of detecting certain immunological
reactions. Proc. Soc. Exper. Biol, and Med., 73: 46-49, 1950.
7. CASTANEDA, M . R., T O V A R , R., AND V E L E Z , R . : Studies on brucellosis in Mexico.
Comparative s t u d y of various diagnostic tests and classification of the isolated bacteria. J . Infect. D i s . , 70: 97-102, 1942.
8. DAMON, S. R., AND ALBRIGHT, K . : T h e isolation of Brucella from blood clots. Symposium on Brucellosis, American Association for the Advancement of Science, Washington, D . C . , 122-125, 1950.
9. E I S E L E , C. W., M C C U L L O U G H , N . B . , AND B E A L , G. A.: Discrepancies in t h e a g g l u t i n a -
tion test for brucellosis as performed with various antigens and as reported from
different laboratories. J . L a b . and Clin. Med., 32: 847-853, 1947.
10. E I S E L E , C. W., M C C U L L O U G H , N . B . , B E A L , G. A., AND B U R R O W S , W . : D e v e l o p m e n t of
Brucella agglutinins in humans following vaccination for cholera. Proc. Soc. Exper.
Biol, and Med., 6 1 : 89-91, 1946.
11. FBINBERG, R . J., AND W R I G H T , G. G . : Factors influencing t h e agglutination titration
in human brucellosis. J . Immunol., 67: 115-122, 1951.
12. F I T C H , C. P., D O N H A M , C R., B I S H O P , L. M . . AND BOYD, W. L . : Studies of t h e test tube
13.
14.
15
16.
17.
18.
agglutination test for t h e diagnosis of Bang's disease (contagious abortion). Technical Bulletin 73, St. P a u l : University of Minnesota Agricultural Experiment Station,
1930. p p . 1-56.
FITCH, C. P., DONHAM, C. R., AND BOYD, W. L . : F u r t h e r studies of t h e t e s t t u b e agglutination test for t h e diagnosis of Bang's disease (contagious abortion). Technical
Bulletin 77, S t . Paul: University of Minnesota Agricultural Experiment Station,
1931. p p . 1-68.
FLEMING, A.: On the influence of temperature on t h e rate of agglutination of bacteria.
Brit. J . Exper. Path., 9 : 231-238, 1928.
GOODPASTURE, E . W., AND ANDERSON, L . : T h e problem of infection as presented by
bacterial invasion of the chorio-allantoic membrane of chick embryos. Am. J . P a t h . ,
13: 149-174, 1937.
G R E E N E , L. F . , AND ALBERS, D . D . : Brucellosis of the urinary t r a c t . Proc. Staff Meet-'
Mayo Clin., 25: 638-640, 1950.
GniFFiTTS, J . J . : Agglutination and an agglutinin blocking property in serums from
known cases of brucellosis. P u b . H e a l t h R e p o r t s , 62: 865-875, 1947.
HALL, W. 11., AND S P I N K . W. W . : T h e r a p y of experimental brucella infection in t h e
developing chick embryo. I . Infection and therapy via the allantoic sac. J . Immunol.,
59:379-391, 1948.
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SPINK
19.'HERROCKS, W. H . , AND K E N N E D Y , J . C..: Mosquitoes as a means of dissemination of
Mediterranean Fever. In Sect, vii, Reports of t h e Commission for the Investigation
of Mediterranean Fever. P a r t I V . London: Harrison a n d Sons, 1906. p p . 70-82.
20. H E S S , W. R., AND R O E P K E , M . H . : A nonspecific Brucella-agglutinating substance in
bovine serum. Proc. Soc. Exper. Biol, and Med., 77: 469-472, 1951.
21. HUDDLESON, I . F . : Brucellosis in M a n and Animals. New York: T h e Commonwealth
F u n d , 1933. p . 231.
22. .JANBON, M., AND BERTRAND, L . : Les adenomegalies de la brucellose humaine. Leur
valeur diagnostique e t pronostique. L a Presse Medicale, 74: 1082-1083, 1949.
23. JANBON, M . , BERTRAND, L., AND QUATREFAGES. H . : L ' a d e n o c u l t u r e . M e t h o d e rapide
et fidele de diagnostic bacteriologique de la brucellose aigue. La Presse Medicale,
77: 1355-1356, 1950.
24. JORDAN, C. F . , AND BORTS, I . H . : Brucellosis and infection caused b v three species of
Brucella. Am. J . Med., 2 : 156-167, 1947.
25. K E N N E D Y , J. C : On the recovery of Micrococcus melitensis from the urine of Mediterranean fever patients. In Sect, iv, Reports of the Commission for t h e Investigation
of Mediterranean Fever, P a r t I I I . London: Harrison a n d Sons, 1905. p p . 56-70.
26. MCCULLOUGH, N . B . : Laboratory tests in brucellosis. Symposium on Brucellosis,
Washington, D . C : American Association for t h e Advancement of Science, 116—
121,1950.
27. M C C U L L O U G H , N . B . . E I S E L E , C. W., AND B E A L , G. A.: Oral a d m i n i s t r a t i o n of killed
• Brucella t o man. P u b . H e a l t h . R e p o r t s , 64: 1613-1616, 1949.
28. M A G O F F I N , R . L., K A B L E R , P . , S P I N K , W. W., AND F L E M I N G . D . : An epidemiologic
s t u d y of brucellosis in Minnesota. P u b . H e a l t h Reports, 64: 1021-1043, 1949.
29. M E T T I E B , S. R., AND K E R R , W. J . : H e p a t i t i s and cholecystitis in the course of Brucella
infection. Arch. I n t . Med., 54: 702-709, 1934.
30. M E Y E R , K . F . : Observations on t h e pathogenesis of u n d u l a n t fever. I n Essays in
Biology. Berkeley and Los Angeles: University of California Press, 1943. p . 437.
31. PICKETT, M'. J., AND N E L S O N , E . L . : Observations on t h e problem of Brucella blood
cultures. J . Bact., 6 1 : 229-237, 1951.
32. POLDING, J'. B . : Some peculiarities in t h e germination of Brucella. J . Comp. P a t h .
and Exper. T h e r a p . , 56: 215, 1946.
33. SCHUBERT, J . H . , AND HERNDON, J . F . : A s t u d y of certain factors affecting t h e agglutination test for brucellosis. P u b . H e a l t h R e p o r t s , 65: 883-890, 1950.
34. SCHUHARDT, V. T . , R O D E , L . J., F O S T E R , J . W., AND O G L E S B Y , G . : An a n t i - B r u c e l l a
factor in peptones. J . Bact., 57: 1-8, 1949.
35. SCHUHARDT, V. T . , W O O D F I N , H . W., AND K N O L L E , K. C . : A heat-labile Brucella-agglu-
tinin-blocking factor in human sera. J . Bact., 6 1 : 299-303, 1951.
36. SHAW, E . A . : F u r t h e r observations on goats, cats, r a t s a n d a m b u l a t o r y peases in connection with Mediterranean fever. I n Sect, iii, Reports of t h e Commission for t h e
Investigation of Mediterranean Fever, P a r t V. London: Harrison a n d Sons, 1907,
pp. 37-42.
37. S P I N K , W. W., AND ANDERSON, D . : Brucella studies on bank blood in a general hospital.
J. L a b . a n d Clin. Med., 35: 440-445, 1950.
38. S P I N K , W. W., AND ANDERSON, D . : Studies relating t o t h e differential diagnosis of
brucellosis a n d infectious mononucleosis: Clinical, hematologic and serologic observations. T r a n s . Assn. Am. Physicians, 64: 428-434,1951.
39. S P I N K , W. W., AND H A L L , W. H . : Encephalomeningitis due t o Brucella a b o r t u s . T r a n s .
American Clinical and Climatological Association, 6 1 : 121-140, 1949.
40. S P I N K , W. W., H O F F B A U E R , F . W., W A L K E R , W. W., AND G R E E N , R . A.: H i s t o p a t h o l o g y
of the liver in human brucellosis. J . L a b . and Clin. Med., 34: 40-58, 1949.
41. SUNDBERG, R. D . , AND S P I N K , W. W . : T h e histopathology of lesions in t h e bone marrow
of patients having active brucellosis. Blood, Supp. N o . 1, 7-32, 1947.
42. W E E D , L. A., AND D A H L E N , D . C : Bacteriologic examination of tissue removed for
biopsy. Am. J . Clin. P a t h . , 20: 116-132, 1950.
43. W E S T , D . E . , AND BORMAN, E . K . : T h e culturing of blood clots for Brucella organisms.
J . Infect. D i s . , 77: 187-192, 1945.
44. W R I G H T , A. E . : Note on t h e technique of serum diagnoses of acute specific fevers.
Lancet, 1: 139, 1897.
45. ZINTEK, A. R . : Brucellosis in Wisconsin. Wisconsin M . J . , 49: 205-208, 1950.