Table Of ContentAN IMMUNOLOGIC STUDY OP HEMAGGLUTININ PRODUCTION
IN TUBERCULOUS AND NORMAL RABBITS
D isse rta tio n
Presented In P a rtia l F u lfillm e n t of the Requirements fo r the
Degree Doctor of Philosophy In the Graduate School of
The Ohio S tate U niversity
By
CHARLES WAYNE DEWITT, JR ., B .S ., M.Sc.
The Ohio S tate U n iv ersity
1952
Approved by:
f Adviser
ACKNOWLEDGEMENT
The author wishes to thank Dr. Jorgen M. Birkeland
and D.r. Matt. C. Dodd for their valuable assistance and
guidance during this study. The author is also grateful
to The Ohio Tuberculosis and Health Association and the
Department of Bacteriology for financial assistance
during the greater part of this work.
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£09397
TABLE OF CONTENTS
Page
In tro d u ctio n ...................................... 1
Review of L iterature , ............................................................................. 5
Materials and Methods ............................................................................. 13
Experimental ............................................................................................. 23
Discussion ........................................................................................... 86
Summary ....................................................................... 914-
Bibliography ............ 96
Autobiography ................................................................... 106
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AN IMMUNOLOGIC STUDY OF HEMAGGLUTININ PRODUCTION
IN TUBERCULOUS AND NORMAL RABBITS
INTRODUCTION
There is a need for a serologic test which will d is
criminate between quiescent tuberculous infection and the
progressive disease, as well as one tLiat w ill distinguish
tuberculosis from diseases that may resemble it pathologi
cally in animals and in man. Such a te st would be useful
not only as a diagnostic tool but would also be a valuable
adjunct in following the course of the disease.
Early attempts at serologic identification of tuber
culosis were far from specific. Bacillary agglutination
(Koch, 19ul) as well as complement fixation (Bordet and
Gengou, 1903; Much, 1912; Harris and Lanford, 1913; Bang
and Anderson, 1913; Negre and Boquet, 1920) gave cross re
actions not only with other species of Mycobacterium but
also with syphilitic sera (Besredka and Jupiile, 1913) and
with sarcoidosis (Carnes and Raff el, 19l|9). The place of
complement fixation in the serologic investigation of tuber
culosis is excellently reviewed by Wadsworth, Maltaner and
Maltaner (1925). Agglutinin adsorption and subsequent bacil
lary agglutination is useful for the differentiation of species
within the genus but too unwieldy for routine work (Wilson,
1925; Furth, 1926). The same is true of quantitative
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complement fixation (Cooke, 1919)*
The precipitin test was trie d in early investigations
and the resu lts are, of necessity, dependent on the antigen
employed. F iltra te antigens such as those of Besredka and
Jupille (1913) and Petroff (1917» 1916) and saline extrac
tions such as those of M iller and Zinsser (1916) showed some
early promise of specificity but did not survive extensive
clinical tests. Seibert (1936) improved the specificity of
precipitation but her high protein antigen gave a low de
gree of sensitiv ity . It is generally accepted that the pres
ence of precipitins is not correlated with the state of the
disease (Rich, I 9I4I4) * Results of sim ilar studies with pre
cipitation of Old Tuberculin fractions seemed to correlate
with the degree of skin sen sitiv ity rather than with the
stage or severity of the infection (Meuther and Macdonald,
19M-1 i McCarter and Watson, I 9I4.2 ). More recent work by Chou-
croun (19)4.7, 1914.7a, 19l|9 ) has shown that the presence of an
anti-carbohydrate precipitin in the serum is related to in
fection and recovery but the very mechanics of precipitation
preclude any great degree of sen sitiv ity .
Workers in other field s, realizing the great difference
in sensitiv ity between agglutination and precipitation, have
made attempts to enhance the sen sitiv ity by increasing the
volume of a precipitate by adsorbing antigens to bacterial
cells (Jones, 1927 > 1926; Roberts and Jones, 19)41; Meuther
and Macdonald, 19ipS) and collodion p articles (Cannon and
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Marshall, 19ij.O; Lowell, 191+3) • The method of collodion par
tic le agglutination was adapted to the investigation of tu
berculous serum by Weir (191+1) but again the specificity of
the reaction depends on the specificity of the antigen chosen
for coating of the particle.
The use of red blood cells as antigen carriers by Bur
net (19lj.6) in the detection of antibodies to mumps virus,
and by Keogh, North and Warburton (19i|8) in the adsorption
of bacterial polysaccharides, led to the idea of antigen se
lectiv ity of ’’schleppers". This was followed by the demon
stration by Middlebrook and Dubos (19^8) that sheep red cells
would adsorb antigen from extracts of tubercle bacixli and
were subsequently agglutinable by specific antiserum. Numer
ous clin ical investigators have designed tests to evaluate
the Middlebrook-Dubos hemagglutination reaction and have ob
tained widely differing results, and offered different in
terpretations of these resu lts. All are agreed, however,
that there is more than one antigen and more than one a n ti
body involved. Following the use by us (DeWitt, 1951J De-
Witt _et _al, 193>1) of this test in the attempted diagnosis
of tuberculosis in c attle , i t was concluded that (1) neither
hemagglutination nor hemolysis are sufficiently specific for
such diagnostic work but (2) that an investigation of the
antigens and antibodies involved would be worthwhile. Ac
cordingly, the following experiments were designed with the
purpose of distinguishing and evaluating those antibodies
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which could be experimentally produced without the presence
of the disease its e lf and, thus, with the hope of removing
specific interfering factors.
REVIEW OF LITERATURE
The agglutinability of red cells following contact with
bacteria or bacterial products was f i r s t recorded by Thomsen
(1927) and attributed to enzymic a ctiv ity by Friedenreich in
1928. The observation that bacteria can a lter erythrocytes
and render them panagglutinabie by most normal sera has been
repeatedly confirmed (Landsteiner, 191+5) • Burnet, McCrea
and Stone (19A4.6) attributed a sim ilar effect by Newcastle
Disease virus to a lecithinase and showed that virus tre a t
ment prevented la te r bacterial adsorption to the same red
cells. Such virus modified red cells were not specific In
their agglutinability (Burnet and Anderson, 19lj-6) but la te r
Burnet (I9I4.6 ), in order to explain the immunizing capacity
of mumps virus treated cells, fe lt i t necessary to assume
that either the virus p article or a breakdown product of
the virus was attached to the red c e ll. Burnet and Ander
son (I9I4.7 ) while working with the "T" antigen of guinea pig
and human red cells showed that Vibrio cholerae extracts
were active in producing this change in the erythrocyte,
and that the enzyme involved could be eluted from the cell,
leaving a previously undetectable antigen on the cell.
The f i r s t definite demonstration of bacterial antigen
adsorption by red blood cells was that of Keogh, North and
Warburton (19i|-8) who used an antigenic fraction which was
composed predominantly of polysaccharides. This phenomenon
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was extended to work with trypanosomiasis by Muniz (1950),
to glanders by Boyden (195b), to tularemia by Alexander,
Wright and Baldwin (195b) and Wright and Feinberg (1952)
and to pertussis by pillemer (195b). Such hemagglutination
was used for investigation of the antigenic relationships
of Mycobacteria by Fisher (1951)* The cross reaction of
tuberculin treated erythrocytes and serum from patients
with Hansen’s disease was deemed diagnostically significant
by Levine (1951),
Middlebrook and Dubos (19ij-h) f i r s t showed agglutina
tion of tuberculin treated red cells in the presence of tu
berculous antiserum. Their primary antigen was a phenol and
acetone insoluble, methanol soluble extract of Mycobacterium
tuberculosis var. hominis (H37Rv) with the activity seeming
ly residing in the polysaccharide fraction. As l i t t l e as
0.ip gamma of antigen per ml could be detected by hemaggluti
nation inhibition, and i t was shown that the antigen was
also present in at least one sample of Old Tuberculin. No
cross reactions were noted with antisera against piplococcus
pneumoniae (I, III, XIV), K lebsiella pneumoniae (B. C) or
serum from sy p h ilitics. The specificity of the reaction was
reported to be good with only occasional reactions of low
titre occurring with serum from patients not actively tuber-
culous.
This in itia l report was soon followed by modifications
and clinical application. Gernez-Rieux and Tacquet (191+9)
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used human group "0" cells as antigen carriers and reported
positive hemagglutination in 88 per cent of patients shown
to have active tuberculosis. Sohier (195U) noted a tenden
cy for well advanced cases of pulmonary tuberculosis to give
negative reactions. This phenomenon is commonly encountered
in other chronic diseases. He reported few false posi
tives except in the case of BCG vaccinated individuals and
later (Sohier, Trimberger and Juiliard, 195b) modified the
test to the use of a purified tuberculin (I.P.lj.8) and human
group "0" cells. Rothbard, Dooneief and Hite (195^), inde
pendently of Sohier, reported the use of tuberculin for cell
sensitization, and lis te d positive results in 5 per cent of
normal humans tested, 18 per cent in other diseases, 6 per
cent in skin test negative individuals and 89 per cent in
proven cases of tuberculosis. Scott and Smith (195u ) pub
lished a simple modification of the reaction which was stan
dardized to the use of a concentrated tuberculin (Lederle,
IpC Int. Std.J, which fa c ilita te d wide-spread comparison of
clinical results. While they at f ir s t presented results
showing hemagglutination titre s greater than 1:2 in 78 per
cent of tuberculous patients, and 35 per cent of BCG vacci
nated individuals and completely negative results in normal
patients, they la te r reported (Smith and Scott, 195^) less
dramatic resu lts. With a larger sampling the percentage of
positive tests in tuberculous individuals changed l it t le , as
did the resu lts in healthy and sick persons with negative
