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THE PERSISTENCE OF THE VI ANTIGEN OF SALMONELLA TYPHOSA IN SOIL AND WATER

 

By
Sarah T. Wade

AiTHESIS
Submitted to the school of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF' SCIENCE

Department of Bacteriology and Public Health

1950

1145.815

AC KNCQ'JLEDGEEN

The author wishes to express sincere appreciation to Dr. H. J.
Stafseth, Professor and Head of the Department of Bacteriology and
Public Health for his advise and counsel, and to Dr.'fl. N. Mack,
Assistant Professor of Bacteriology, for his supervision and guidance.

The author also wishes to express sincere appreciation to Dr. W. F.
Ferguson, Director of the Salmonella Laboratory, Michigan Department of
Health for supplying the cultures and phages and for his criticisms of
this thesis, and to Mr. Warren Litsky, Department of Bacteriology, Mich-

igan State College for his helpful suggestions.

TABLE OF CONTENTS

 

 

 

 

 

 

 

Introduction 1
History ~ A
Materials 9
Methods 12
Discussion and results 36
Summary 39
Bibliography 40

INTRODUCTION

For many centuries typhoid fever has been one of the most dreaded
and deadly diseases of mankind. It is now an uncommon disease in this
countey, but it would be wrong to assume that it is already in the pro-
cess of extinction. Endemic loci of the infection, maintained by chronic
carriers, still exist in many parts of the country. Only one death from
typhoid fever was reported in Michigan during 1949, however, there were
forty-eight recognized and reported cases according to Leeder (1950).

The control of typhoid fever depends ultimately on the detection
and control of the chronic carrier. The detection of a carrier is usuah—
1y a difficult task, however, the new bacteriophage typing method devised
by Craigie and Yen (1938) has provided a reliable guide to the epidemic-
logical investigation of outbreaks or sporadic cases of typhoid fever.
Its application greatly facilitates the detection of chronic carriers,
especially those responsible for sporadic cases in endemic areas.

The bacteriophage typing method reveals strain differences which can-
not be demonstrated by any serological method. Because these strain
differences remain unaltered on human passage, they provide a means of
identification whereby transmission in man may be traced. A carrier may
by absolved from.responsibility for a certain outbreak if he is excret-
ing a strain of Salmonella typhosa which differs in Vi-bacteriophage type
from.those isolated from the diseased cases. The bacteriophage typing
method permits the epidemiologist to trace the connections between spor-
adic cases of the disease. This is a task which cannot be accomplished
without the use of this new laboratory aid.

1

In the past, the major epidemics have been caused by contaminated
food or polluted water, the carrier being the indirect source of the
organism. Even though improvement in the proper disposal of sewage has
greatly helped to improve conditions, the rural areas in many sections of
the world are still sources of danger.

If the fecal material is deposited on the soil or used as fertilizer,
the soil may serve as a medium upon which the organisms may survive.
Vegetables grown on the infected soil may harbor the organisms and when
the vegetables are marketed, the typhoid bacillus may serve as the source
of a new outbreak of the disease. Wells, rivers, and streams, which are
polluted with'S. typhosa may also serve as the source of infection. A
number of studies have been carried out on the longevity of the typhoid
bacillus in soil and water, but as Beard (1940) has pointed out, the re-

sults Show no close correlation.

Felix and Ritt (l93h) were the first to recognize that the type of
Ԥ. typhosa which occurs in human infection is characterized by antigens
'which are distinct from.the O and H antigens, namely the Vi antigen.

Since the Vi antigen of‘S. typhosa is present, practically without
exception, in cultures isolated from.patients with typhoid, Felix and
others have reasoned that the Vi antigen is intimately associated with
virulence or invasive ability. This theory has been substantiated by
animal tests and by observations ig,zitgg, whenin it has been shown that
loss of Vi antigen is one of the first steps in the degradation of a cul-
ture. It is noteworthy that W forms ofl§. typhosa are found with con-
siderable frequency in old carriers in whom, it has been conjectured, the
immune forces which hold the organism in check bring about the V W changes.

2

Because the presence of Vi antigen is so closely related to viru-
lence, a test for the antigenic component constitutes a rough qualita-
tive method of determining virulence. In the present study an attempt
has been made to determine the persistence of Vi antigen in typhoid
organisms inoculated into soil and water. It is felt that experimental
data resulting from.this study will be of value both to the sanitary
engineer and to the epidemiologist.

The method used to determine the presence of the Vi antigen on the
organisms from soil and water was that of bacteriOphage typing, which

was described by Craigie and Yen in their original work in 1938.

HISTORY

During the past twenty years considerable new knowledge has been
accumulated concerning the antigenic structure and antigenic variations
of‘S. typhosa. Until 1930, it was generally accepted that the typhoid
bacillus consisted of a somatic and flagellar antigens, but few, if any
theories had been advanced to explain the relationship of virulence and
resistance to 0 agglutination by 0 antiserwm.

In 193A, Felix and Pitt published a report of their work which re-
vealed that virulence was closely associated with resistance to the ac-
tion of the 0 antibody. They were the first to show that the inagglutin-
able strains were highly virulent, whereas, agglutinable strains were
less virulent. Both types of the organism had the general characteristics
of smoothness and did not differ in their content of smooth 0 antigen.
This indicated that the mere presence of smooth 0 antigen did not of it-
self determine high virulence, but that some unknown factor was required
to render this antigen resistant to the action of the 0 antibody.

In a later report, during the same year, Felix and Pitt (l93h) con-
cluded that virulence and resistance to O agglutination by 0 antiserum
were correlated by the presence of an antigen, separate and distinct from
the long-established O and H antigens. They called the newly discovered
antigen the Vi antigen or Virulence antigen.

Kauffmann, in 1935, made an extensive study of the typhoid bacillus
primarily from the point of view of the agglutination reactions and par-
ticularly the presence of the Vi antigen. He introduced the terms "V form"

and "W form" to denote, respectively, the form of‘S. typhosa which contained

h

the Vi antigen and the form which did not. An crganism.with the Vi anti-
gen was agglutinated by the pure anti-Vi serum but not by a pure anti-O
serum. Those organisms which did not ShOW’the presence of the Vi antigen
in agglutination reactions were classified as "W form", that is, organisms
which were not agglutinated by anti-Vi serum, but were agglutinated by
the anti-O serum. Kauffmann found that the organism.may also exist in an
intermediate form. The intermediate form showed partial agglutination in
both a pure anti-Vi and a pure anti-O serum, and thus he called it the
"VAW form".

Craigie and Brandon, in 1936, found that certain strains of bacterio-
phage would lyse the typhoid bacteria bearing the Vi antigen. This served
as the foundation which lead to the typing of §, typhosa by bacteriophage.

In 1938 Craigie and Yen isolated four Vi-specific bacteriophages.
They observed that one of these bacteriophages had the unusual ability to
adapt itself to certain strains oflfi. typhosa containing the Vi antigen.
Realizing the highly selective, lytic activity that bacteriophage type II
was capable of developing, Craigie and Yen (1938) set up a series of ex-
periments in which they conditioned the bacteriophage to particular strains
of §, typhosa. Rawlings, Watson, and Ty2 were the first typical strains
studied. Bacteriophage type II was propagated on the Rawlings and watson
strains and the bacteriophage developed an equally high lytic activity for
both. When the bacteriophage, grown on these two strains, was applied to
Ty2, it was found that it had lost its lytic ability except in very high

concentrations. The original bacteriophage type II was then applied to

strain Ty2. It was observed that lysis occured, and that the bacteriophage

5

had developed a high specificity for the Ty2 strain and lost its ability

to lyse the Rawlings and'flatscn strains. Several preparations of bacterio-
phage, showing selective affinity, were made by adapting the bacteriophage
type II to different strains ofԤ. typhosa.

Craigie and Yen (1938) made the supposition that there are different
varieties of S, typhosa which differ only in their Vi antigen and the
variations or differences in the Vi antigen could be shown only by the
action of adapted bacteriophage type II.

This meant that the bacteriophage appeared as a number of mutants,,
differing in their affinity for different types of typhoid bacilli.
Differences in the strains caused differences in environment which favorb
ed mutation. The dominant mutants continued to propagate and the others
did not. By growing the mutant on the strain capable of supporting it,
which is really selective propagation of the mutant, a series of phages
were obtained showing a high relative affinity for the homologous strain
of S, typhosa. Eleven phages were originally produced by this method of
adaption and selective propagation and since that time the number has
increased to twenty-five known types.

The eleven original phages were designated as A, B1, Bz, C, D1, D2,
E, F, G, H and L. The letter "I" was intentionally omitted in the desig-
nation of types to avoid possible confusion with Phage Type I. The comp
plete list of known types and subtypes include A, B1, B2, 83’ C, D1, D2,
DAHP56’ D5, D6, E1, E2, F1, F2, G, H, J, K, L1, L2, M, N, O and T. The
reaction of these phages when tested against the corresponding series of

Vi type strains of‘S. typhosa are given in the following Table I.
6

THE FOUR ORIGINAL VI PHAGES

 

 

 

on which it is 1

 

 

Vi Phage Relative Thermal Neutralization Lytic activity
particle Death by antiphage for V forms of
Size Point serum S, typhosa
I II III IV
Type I Large phage 67-7000. Lyses all V forms
Type II medium phage 69-7200. Develops a highly
selective lytic
activity for the
type of‘S. typhosa
propagated.
Type III Small phage 61-64°C. Lyses majority
of V forms
Type IV Medium.phage 59-6200. Lyses dajority

of V forms with
exceptions of
type F and D1.

 

From the data in this table, Craigie and Yen.Cl938) concluded that:

l. The four phages are specific for the V form of‘S. typhosa.

2. Serologically, the four types of phage are distinct.

3. A variation occurs in the lytic action on V form.strain of‘S. t hosa,

particularly in the case of Type II phage.

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8

MATERIALS

The twenty-two standard Vi phage preparations used in this study
were obtained in concentrated form from the Laboratories of the Michigan
State Department of Health, Lansing, fiichigan. Originally they were ob-
tained from the Connaught Medical Research Laboratories, University of
Toronto, Toronto, Canada. The phages used included: A, B1, B2, B3, C,
D1, D2, DAHP56’ D5, D6, 31, E2, F1, F2, G, H, J, K, L1, L2, M and N.
Phages O and T were not included in the group. The four Vi-type cultures
of S; typhosa (A, DAHP56, El and F1), used in this work, were supplied
from.the same source.

The soil samples used for determining the persistence of the Vi
antigens were obtained from the Soils Department of Michigan State Cohlege.
The soil samples were of four types, namely: Oshtemo sand, Miami sand,
Brookston clay and muck. By taking four different types of soil, a wider
range in the amount of organic matter present could be obtained. The_
water samples for this study were taken from the Red Cedar River.

The concentrated bacteriophages were diluted to their "Critical Test
Concentration" (C.T.C.) before they were used for the typing. "C.T.C."
is defined as the greatest dilution of bacteriophage that will produce
lysis on the homologous strain of‘S. typhosa. The proper dilution for
the above mentioned bacteriOphages was found to be l-lOO.

The media used for typing were prepared according to the work of

Craigie and Yen (I938).

Agar medium

'Bacto' nutrient broth (dehydrated) 20.00 gms.
Sodium chloride 7.50 gms.
'Bacto' agar ‘ 20.00 gms.
Distilled water to 1000.00 fiml.

Phage dilution broth
'Bacto' nutrient broth (ddhydrated) 8.00 gml.

Distilled water to 1000.00 ml.

Phage culture broth

'Bacto' nutrient broth (dehydrated) 15.00 gms.
Sodium chloride . 7.00 gms.
Distilled water to 1000.00 ml.

Craigie and Felix (19A?) stated that any good meat digest agar may
be used as an alternative medium providing that the standard reactions
are reproducible. The media used in this study, however, were prepared
according to the above mentioned formulae. The desired results could
not be obtained by substituting different agar.

'Bacto' tryptose agar was used for growing the organisms to seed the
soil and water; saline solution'(.85 percent) was used to wash the organ-
isms from the agar. 'Bacto' tetrathionate broth base was employed as the
selective liquid enrichment medium; HacConkey, 8.8., and bismuth sulfite
as the differential media for the isolation of‘S. t hose, and Kligler's
iron agar slants for further identification of the organisms.

10

The cultures were stored on Dorset Egg medium, which retains the
Vi antigen. The Dorset Egg medium.consists of three parts of whole egg,
one part of distilled water. It was slanted and inspissated. The stock
strains were transferred to fresh slants at four-month intervals follow-

ing passage on nutrient agar and tests for specificity.

METHOD

The four soils, Oshtemo sand, Miami clay, Brookston clay, and muck,
were each divided into four samples of 300 gms. By taking four soils.

a representative sample of various soils was obtained, each differing in
the amount of organic matter present. Moisture content tests were per-
formed on each of the samples at the beginning of the study and the re-
sults obtained are shown in Table II. The optimum water-holding capacity
of the various soils according to Lohnis and Fred (1923) is: Oshtemo sand-
63, Miami clay- 10 to 11%, Brookston clay- 25%, and mmck- varies up to
200%. The moisture content of the samples was adjusted to approximately
that of standard conditions and kept at the optimum throughout the experi-
ment. Moisture content tests were conducted by weighing out 10 gm

samples of the soil, drying until a constant weight (usually LB hours)

was obtained and then calculating the percent of moisture from the loss

in weight.

A sample of each soil was seeded with one strain of‘S. typhosa. The
four strains of'SK typhosa used for the seeding were A, DAHP56’ El’ F1.
This made a total of 16 jars. Ordinary quart fruit jars were employed.
They were covered with Mason lids, but not sealed air tight. The temper-
ature was that of the room in which they were kept. (approximately 22°C.)

Before seeding the soil with the organisms, a 1:10 dilution of soil
in tetrathionate broth was made and incubated for twenty-four hours. From
the tetrathionate broth, plates of bismuth sulfite, EacConkey's and 3.8.
agar were streaked. A few colonies which might be confused with those
of S, typhosa were picked, stabbed into and streaked on Kligler's iron agar
slants to verify the fact that no salmonella-like organisms were present.

12.

 

 

 

 

 

 

 

Table II
OSHTEZJO SAND
Typhoid Wt. of the flask 3%. of the flask %Moisture
types 10 gm. of soil soil after drying
before drying at 115°C.
A 31.7 gm. 30-8 gm. 9%
E E 32.0 gm. 31.01 gm. 9.9%
F 32.0 gm. 30.30 gm. 7.9%
MIAMI SAND
A 31.2 gm. 29.6 gm. 1676
134mg6 31.8 gm. 30.1. gm. 11.55
E 31.5 gm. 30.0 em. 14%
F 31.7 gm. 330.3 gm. 14%
BROOKSTON CLAY
A 32-0 gm. 30.35 gm. 16.5%
0’
DAHP56 31.2 gm. 29.5 gm. 17/6
E 31.0 gm. 29.2 gm. 18%
F 31.7 gm. 30.0 gm. 17%
MUCK
A 32.0 gm. 26.3 gm. 56.9%
DAHB56 3107 9110 2603 gm. 43.2%
E1 31.6 gm. 26.1 gm. 55.0%
F1 32.3 gm. 26.7 gm. 56.0%

 

13

in the soil before seeding. The four typhoid strains were typed by
bacteriophage before seeding the soil to make sure the Vi antigen was
present.

8 The organisms to be seeded were taken from the stock cultures,
broth suspensions made, incubated for twenty—four hours, plated in Kolle
flasks containing tryptose agar, incubated for twenty four hours and
then washed from.the agar with .85 percent saline solution. The saline
suspension of the organisms was used to seed the soil. This suspension
contained some nutrient which would be added to the soil and water, and
which might help to maintain the organisms in the samples.

Five-tenths m1. of an .85 percent saline suspension of the organism
was added to 30.00 m1. of distilled water and this was added to the 300.00
gm. of soil for each of the sixteen samples. By adding a 30.00 m1. sus-
pension of the organisms, the organisms could be more uniformly distri—
buted throughout the sample.

At weekly intervals, a 2 gm. sample of soil was taken from the re-
spective jars and tested for longevity of the typhoid bacillus and the
presence of the Vi antigen by bacteriophage typing. The soil sample was
added to tetrathionate broth, incubated for 12-18 hours, then streaked on
bismuth sulfite, HacConkey's and 3.5. agar and incubated for 48 hours.
Colonies were picked from.the differential media, stabbed into and streaked
on Kligler's iron agar slants and incubated for 24 hours. The character-
istic reaction of'S. typhosa in Kligler's iron agar is acid butt, alkaline
slant, and H28 production. The organisms identified on Kligler's iron agar
were then ready for typing for the presence of the Vi antigen.

1A

The procedure used for typing was essentially the same as described
by Craigie and Yen (1938). A small amount of the growth on Kligler's
iron agar was inoculated into 2 ml. of nutrient broth and the tube incu-
bated at 37°C. for 2 hours. The inoculation sites were then made on nu-
trient agar plates in the following manner: A loopholder, containing a
platinum loop (2.75 mm. internal diameter) was taken between the thumb
and first 2 fingers like a pen and a full and uniform.drop of broth culture
was obtained by quickly breaking the surface of the broth. With the fore-
arm.and side of the hand resting on the table the loop was lowered until
the drop touched the surface of the agar, then the loop was given a hori-
zontal twist by moving the fingers in an area approximately 15 mm. in
diameter. The loop should never touch the agar because it may cause an
area which may be confused with lysis. The loopholder should be used
merely to direct the spread of the culture. After the agar had absorbed
the fluid, usually five to ten minutes, the bacteriophage was applied to
the center of the inoculated area. TWO loopholders may be used alternate-
ly to conserve time. In applying the bacteriophage the loopholder should
be held between the thumb and fingers in a vertical position. The loop-
holder was then lowered until the loop almost touched the surface of the
agar and then rotated in a vertical position and the contents allowed to
flow onto the center of the inoculated area. The bacteriophage was al-
lowed to spread naturally on the sited area. The plated were then inverted
and incubated at 37°C. for two hours and stored in the refrigerator at
4-600. overnight. In the morning the plates were returned to the incu-

bator for four to six.hdurs before reading the results.

15

It is important that the plates never be stacked, because‘g. typhosa
will grow, but will not show lysis below’37°C.I The plates should be pre—
pared at least one day before being used because excess moisture delays
absorption of broth and bacteriophage. The plates, however, should not
be prepared too far in advance because excessive drying causes uneven
distribution of the culture and bacteriophage.

Uninterrupted incubation can be used with success, but the interrupted
method has proved to be more advantageous:

l. The interrupted incubation method permits phage reactions to be ob-
served at their optimum and before they are obscured by late growth.
2. The intermediate period in the cold room permits diffusion of the

phage into the surrounding normal culture.

16

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DISCUSSION OF RESULTS

The results given in Tables III-XX show that §. typhosa survives
in Oshtemo sand, Miami clay, Brookston clay, muck, and water from the
Red Cedar River for varying periods of time. In addition to showing the
survival of the organism, the tables also show the persistence of the
Vi anyigen. This latter point is of special interest since the Vi anti-
gen is related to virulence. Table XXI summarizes the results reported
in the preceeding eighteen tables.

The Oshtemo sand did not support the organism for more than two
weeks and only type DhHfifisurvived for this period. After the first typ-
ing following seeding, type DLHP56 showed only semi-confluent lysis. More
will be said about this type and its reactions later. Types 1 and Fl sur-
vived only one week, but showed equal growth and presence of the Vi anti-
gen. Type E1 did not survive through the first week after seeding into
the soil sample.

Brookston clay provided conditions necessary for survival through
six weeks. Here again type DAHP56 outlived the other types by two weeks,
but it did not show confluent lysis after the first typing which followh
ed seeding. Type E1 survived through the fourth week and the others i.e.
A see F1 survived three weeks but not four. The latter three types showed
good confluent lysis when typed with bacteriophage.

In Miami clay, type A survived through the fifth week, however, dur-
ing the last two weeks of its survival, no clear confluent lysis was ob-
tained. The semi-confluent lysis which was produced indicated that the

organism was losing its Vi antigen. Confluent lysis was obtained only

36

through the third week. Type DLHP56 organism grew through the third week
but was not typable after the first week. Apparently the organism had
completely lost it Vi antigen after this short period. Types E1 and F l
survived and gave confluent lysis through three and four weeks respective-
ly.

The muck soil gave results closely parallel to the Brookston clay.
Type DAHP56 was recovered through six weeks and as in the Brookston clay
sample it showed confluent lysis after seeding the soil. At the end of
one week and until the organism died out, it produced only semi-confluent
lysis. Types A, El, and F1 survived four, three and four weeks respective-
ly; For all three types, the Vi antigen remained as long as the organism
survived.

In the water samples all the typhoid organisms died out at the end
of one week. All fourtypes (A, DAHP56, E1 and F1) gave confluent lysis
until the death of the organism, which indicated that the Vi antigen was
present as long as the organisms remained alive.

Type DLHP56 persisted for a greater period of time in all samples
except the Miami clay and as long as type A in water. In soils where this
type of organism survived the longest, it lost its ability to produce con-
fluent lysis in less than one week. The consistent appearance of semi-
confluent lysis which occurred following the first week indicates that
this type differs from the other types. The specific bacteriophage,
DAHP56’ was tested with the original stock culture of type DAHP56 so as
to make sure that the bacteriophage was not contaminated. Several fact-

ors may be involved which may account for the organism losing its ability

37

to exhibit confluent lysis. It is believed that when the organism.begins
to lose its Vi antigen, it will not show confluent lysis. Another explan-
ation of this phenomenon, as is indicated in Table I, is that, when the
bacteriophage is at its critical test concentration, a cross between
closely related organisms and bacteriOphage may show semi—confluent lysis.
The most likely explanation, however, is that this bacteriophage DAHP56

carries a latent bacteriophage which interferes with the typing.

38

SUMMARY

In this work with four types of soil and one water sample, the
longevity of the four types ofԤ. typhosa and the persistence of their
Vi antigen were determined by using bacteriophage typing technique.

In general, the survival of the organisms in Brookston clay, Kiami
clay and muck, when the three were maintained at their optimum.water-
holding capacity and a temperature of 22°C., varied from three to six
weeks. In Oshtemo sand and in river water the organism did not survive
for three weeks. §, typhosa types A, El , and F1 showed no consistency
in longevity in any of the soil and water samples, but the Vi antigen was

found as long as the organism remained alive.

‘5. typhosa type DAHP56 gave semi-confluent lysis in the four soil
samples used. This type of the organism also survived longer than the
other three types in Oshtemo sand, Brookston clay and muck. Ԥ. txphosa
type DAHP56 showed confluent lysis in all samples in the first typing
after seeding, but after the first typing it produced semi-confluent lysis.

Possible explanations for the appearance of semi-confluent lysis are given

in the discussion.

39

BIBLIOGRAPHY

l. Almon, Lois: The significance of the Vi antigen. Bact. Rev., é:Z:
(19h2-h3): h3-53.

2. Beard, Paul J.: Longevity of Eberthella typhosus in various soils.
Am. J. of Pub. Health, 29, (1940): 1077-1082.

3. Bradley, W. H.: An epidemiological study of Bacterium typhosum.type
DA. Brit. Lied. J., _1_, (1943): 1.38.

 

h. Burnet, F.M.: The relationships between heat-stable agglutinogens
and sensitivity of bacteriophage in the Salmonella group. Brit. J.
of Exp. Path., g, (1927): 121-129.

5. Budd, William: Typhoid fever-its nature, mode of spreading and pre-
vention. George Grady Press, New York; 1931.

6. Craigie, James and Brandon, K.F.: Identification of the V form of
g. typhosu . Can. Pub. Health J., g, (1936): 165-170.

7. CCraigie, James and.¥en, Chun Hui: The demonstration of types of B.
typhosus by means of preparations of type II Vi phage. Can. Pub.
Health J., 2 , (1938): 448.

8. Craigie, James and Felix, A.: Typing of typhoid bacilli with Vi
badteriophage. Lancet, (1947): 823-836.

9. Crocker, Clarice: Craigie Vi phage method and South African strains
of‘B. t hosus, with special reference to typhoid carriers. J. of
Hyg-: 45. (19h?) 118-122.

10. d Herelle, Felix: The bacteriophage and its behavior. Williams and
Wilkens 00., Baltimore: 1922.

11. Felix, A.P Experiences with typing of typhoid bacilli by means of
Vi bacteriophage. Brit. Med. J.,‘l, (1943): LBS-A38.

12. Felix, A. and Pitt, R.M.: Virulence of B. t hosus and resistance
to 0 antibody, J. of Path. and Bacty, 2g, (193A : LO9-h20.

l3. Ferguson, W.: Personal communication.

14. Henderson, N.D. and Ferguson, W.W.: Bacteriophage typing of Salmonella
typhosa. J. of Lab. and Clin. Med., 2&, (l9h9): 739-7L3.

15. Kauffmann, F.: Uber einen neuen serologischen Formenwechsel der
Typhus-bacillen., Zeit. fur Hyg. und Infect. llé, (l93h-5): 617-651.

A0

16.

17.

18.

19.

20.

21.

Lazarus, Alfred 3.: Typhoid typing in the Western States. Am. J.
of Pub. Health., 31, (19A1): 69-6A.

Lazarus, Alfred S. and Gunnison, J.B.: The action of Pasteurella
pestis bacteriophage on strains of Pasteurella, Salmonella, and
Shigellao J. or BaCtyo, é, (191(7): 530

Leeder, F.S.: Personal communication.

Lohnis, F. and Fred, E.B.: Textboofl of Agricultural Bacteriology.
McGraw Hill Book Co., Inc. New York: 1923.

Morris,J.; Brim,A., and Sellers, T.F.: Types of Eberthella typhosa
found in Georgia during the four year period l9Al-AA. J. Infect. Dis.,
21’ (1945) 3 25 o

Mbrris, J.F. Brim, A., and Sellers, T.F.: An outbreak of typhoid
fever due to the small colony variety of Eberthella typhosa. Am. J.

_ of Pub. Health., 22, (1943), 246—248.

Polson, A. and wyckoff, Ralph: The amino acid content of bacterio-

phage. Sci., lgg, (19A8): 501.

23.

flyckoff, Ralph W.G.: Symmetrical Patterns of Bacteriophage production.
Proc. of the Soc. for Exp. Bio. and Med.,‘éé, (l9h7)? A2-AA.

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