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A STUDY (1“ THE CLOUDY REACTICNS IN THE AGGLUTINATIQI TEST

FOR SALMONELLA PULLORUM INFECTICN

*****#**¥*

A Theeie Presented for the
Degree of Master of Science

By

George S. Schilling, B. S.

******##**

Michigan State College

1927

**********

THESIS

 

A

INTRGDUCTIG

In 1899, Rettger isolated the causative agent of bacillary white

 

diarrhea of chicks, Salmonella 111110331121, w’BfierimJullorg as it was
formerly designated. Rettger and Stoneburn in 1909 determined that the
original source of the infection is the hen, that the organism is contained
in the yolks of eggs fran infected hens, and that chicks produced from in-
fected eggs may have the disease when hatched. These findings served to
focus attention on the carrier problem, and Jones adapted the agglutination

test to the identification of adult birds harboring Salmonella pullorun

 

infection. Since that time large numbers of birds have been tested in
various states of this country in an effort toward the suppression of bacil-
lary white diarrhea.

me of the difficulties encountered early in the testing for
S. pullorun infection by means of the agglutination test was that a non-
specific precipitation frequently appeared when chicken serul was added to
the test fluid, and interfered with a proper reading of the results of the
test. This non-specific precipitation has been spoken of in the literature
as a 'cloudy reaction" and in want of a more convenient term will also be
used in this paper. Some studies have appeared heretofore which were design-
ed to throw light on the reason for the occurrence of this phenanenon or to
find a means of preventing it; these 1:111 be referred to later in connection
with the presentation of further work on the same problem. An entirely
satisfactory means of preventing the appearance of cloudy reactions does
not appear to have been developed, and the explanation of the reason for
its occurrence also appears to be wanting. The study here presented was
undertaken to find the cause of, and to find a means of inhibiting such

non-specific precipitations. 103523

In conducting this work, materials were secured in conjunction
with the application of the routine diagnostic procedure on the serum of
over 3400 birds» In presenting the findings, it will be convenient to
discuss the different means of approach and the information secured in
separate sections. Thus, this paper presents the following divisions:

l. The incidence of clouding reactions as encountered in
routine diagnostic tests on standard antigen.

2. The chemical nature of. the clouding factor.

3. The source of the precipitating material.

4. The investigations on means of preventing the precipitation
of substances responsible for clouding.

6. The bactericlcgic and pathologic findings on birds giving

cloudy, or positive reactions to the agglutination test for S. pullorum

 

infection.

The fifth section does not relate directly to the problem of
explaining or inhibiting cloudy reactions. The studies on bacteriological
and pathological findings were made however in conjunction with the studies
on cloudy reactions; they throw light on the accuracy of the diagnostic
work performed, and on the differentiations here made between cloudy and

positive reactions. For this reason they are also included.

 

Iu'l'hese investigations were undertaken in the laboratories of the Depts.

of Vet. Science, and Bacteriology, Arkansas State Experiment Station.

 

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,1- : Hun puma-M e4

SECTICH I
THE INCIDENCE G‘ CLWDY REACTIQIS AS ENCOUNTERED DI RGITINE DIAGNOSTIC
TESTS ON STANDARD ANTIGEN
The cloudy reaction occurs frequently in diagnostic tests for

carriers of _8__._pullcrum and makes it difficult or impossible to determine

 

whether or not agglutination has also occurred, or whether the serum which
produced the cloudy reaction was obtained fro an infected bird. Mathews-1-
found that as high as 75% of the birds of acne flocks may give the cloudy
reactim. Taking into account the possibility that a cloudy reaction may
mask (or may prevent?) the appearance of the agglutination reaction the
comittee on Poultry Diseases of the U. 8. Live Stock Sanitary Association-2-
has recommended that all birds giving a cloudy reaction be condemnedunless '
retested. Thus, it is quite apparent that the interference of this non-
specific precipitation is a matter of serious consequence.

A number of influences such as age and sex «1’ the birds, season
of year, and that of a period of starvation preceding the taking of blood
samples have been correlated with the occurrence of cloudy reactions. Follow-
ing the work of Warner and Edmond-:2 who showed that the blood of older birds
does not contain as much fat as that of pullets, Hitchnerg tried the prac-
tice of starving the birds 36 hours before bleeding; this he found eliminated
part of the interference. Stafsethg made the observation that the cloudy
reaction is more commonly observed with the blood of pullets, and his work
also showed that starvation prior to bleeding reduces the number of samples
showing cloudy reactions, although the type of ration which the birds re-
ceived appeared to be without influenoeas. He further found that fewer
cloudy reactions are encountered in October, Novenber, and December, and

he did not encounter this reaction with the blood of male birds.

 

, a r

 

 

will!

7‘

That clouding may be predicted by noting the appearance of chicken
serum.at the time of setting up the test has been suggested by'Matheweéw
This investigator further states that by examining a quantity of samples of
blood serum one may select those which may be expected to yield the cloudy
reaction and he recommends using such sera with antigen containing sodium
hydroxide.

In this work, blood samples which had been submitted to this labo-
ratcry were utilized, the source of blood samples, the sex of birds, the
breeds represented, and the condition of the serum'were recorded. The samples
were used in the routine diagnostic tests for bacillary white diarrhea by the
agglutination method; and cloudy reactions as well as positive specific re-
actions with their relative time of appearance were entered separately into
permanent records. This data thus became available for a study to deter-
mine whether correlations exist as.between the factors mentioned and the
appearance of the cloudy reaction. Supplementary tests related to the
relativeltime of appearance of the agglutination reaction and of the cloudy
reaction.

Experimental Procedure

The antigen employed in this work was made by cultivating five
strains of S. ,pullorum separately on standard agar. In 48 hours when approx-
imately maximum growth had occurred, the cultures‘were'washed off with physio-
logic saline solution containing 0.5% phenol. They were combined into one
composite suspension of organisms, filtered through glaes'wocl, and heated
at 60° 0 for 30 minutes. The casposite suspension was placed in refrigera-
tion, and as needed portions were removed and diluted with phenolised saline
so that the turbidity was reduced to be canparable with tube one of the
McFarland nephelometer; the dilutions thus secured from the composite sus-

pension served as the antigen.

The strains of Si. pullorum used were examined by cultural and

 

microscopic‘methods to determine the purity of culture and conformity to
type. They were found to be characteristic; the carbohydrate fermenta-
tion reactions of the strains are appended in tabular form. Table No. l.
Tests for acid and gas production on Dulcitol, Lactose, Maltese, and

Sucrose were also made which were uniformlynegative. Details of prepar-

ation of the carbohydrate media may be found in Section V of this paper.

Table No. 1

Fermentation Reactions by Strains of S. pullerum used in the
Standard Antigen.

 

 

Straw: Arab inose : Dextrose : Galactofi: LeFuTose :Mannitcolzllannose : Rhemnose :Xonsg:
:Acid: Gas:Acid:Gas:Acid;Gas:Acideas:Acid:Gas:AcidGas:Acid:Gas:Acideas

 

 

$4 a GA -A G AGA G AG AG.--
2142 A CA GA G AGA G AG A-A-
a A -A -A G AGA G AG AG--
P A GA GA G AG- - AG AG--
K A -A -A - --- - AG A---

 

. The first three strains were furnished by courtesy of Dr. B. A. Beach of
the Wisconsin Experiment Station. The other two by Dr. R. E. Rebrassier
of the College of Vet. Medicine, Ohio State University.

The blood samples were obtained from sixteen different flocks.

They were drawn into 8 by 75 mm. tubes; all tubes were boiled with clean-

ing solution, thoroughly rinsed, dried, corked with bark stoppers, and

sterilised prior to use. Upon the arrival of the blood samples at the labo-
ratory, the clots were loosened from the walls of the tube, and the serum
separated from the clot by centrifugation. After setting up the tests, the
blood samples were held under refrigeration for 72 hours to afford oppor-

tunity to observe changes in the whole blood serum which might occur.

In setting up the tests one mil of the antigen was placed in
each of the 8 by 76 m.m. test tubes. Each serum was set up in two dilu-
tions, 1 to 50 and l to 100 by placing respectively 0.02 and 0.01 mile of
serum in each of two tubes. The l to 50 dilution however was used as the
criterion for charting positive specific reactions, or the non-specific
cloudy reactions. The test tubes used in setting up tests were regularly
cleaned before using in the same manner as described for the tubes in
which blood samples were collected.

Thorough mixing of serum and antigen was secured by shaking
each tube when the blood serum was introduced. The tubes were then in-
cubated at 37° 0. Except when specifically stated to the contrary final

readings were made at 48 hours.

Time of Appearance of Clouding and Agglutination Reactions

In a group of 834 acre readings of the agglutination test were
made at the end of two, twelve, twentybfour, as well as at forty-eight
hours incubation. The object here was to determine the relative time of
appearance of the specific and the cloudy reaction, and to find the most
favorable incubation time at which to read the tests for purely diagnostic
purposes, i.e., the length of incubation are least apt to be present and
mask or inhibit agglutination.

The tabulated results are given in Table No. 2. It should be
noted that of the tubes which showed clouding at 2 hours, a considerable
number became cloudy immediately after the addition of the serum, and that
others gave a prompt reaction by showing clouding prior to»being placed
to incubate while standing at room temperature.

The data clearly show that the cloudy reaction tends to appear

earlier than the agglutination reaction. Since this is the case, and

-e-

since the cloudy reaction when once it has appeared tends to persist, a
' selection of the time of reading does not offer a means of escape from

the interference of this non-specific precipitation.

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Condition of Serum as Related to Cloudingg

 

The interesting statement by Mathews}- that cloudy reactions can
be predicted by the appearance of the serum and that taking the precaution
of adding sodium hydroxide to the antigen in which such sera as give visu-
al evidence of being likely to cloud are to be set up, is a means of pre-
venting interference, led to the compilation of data on the point whether
a serum which varied from the normal clear straw color invariably clouds,
and whether such a normal serma ever produces cloudy reactions.

In order'not to make the divisions of the degree of precipita-
tion and alteration fraa the normal clear scrim so fine that a multiplicity
of designations might lead to confusion as well as not be significant,
three designations were adopted, namely, clear serum, i.e., a clear serum
of a pale straw color, roily sermn, i.e., a serum opalescent to milky in
appearance, but which did not contain aggregations of suspended precipi-
tates, and precipitated serum, i.e., a serum having definite precipitates
and giving a curded appearance, whether the precipitate was floating as
a layer at the top of the tube, or was suspended in the sermn, or had
settled out. in the tube.

In Table No. 3 are presented the tabulated results on five dif-
ferent lots of sera, comprising a total of 1013 samples. For purposes of
this comparison, in no case were sera showing laking considered.

Discussion: An inspection of the table shows that relatively
the percentages of clear and precipitated sera which cloud in antigen are
about equal. 01‘ the roily sera, the chances are about equal that such
sera will produce clouding or remain clear in the agglutination system.
For of 911 clear sera, 323 or 35.4% produced clouding, of 66 precipitated
sera 17, or 30.3% produced clouding, and of the 46 roily sera, 26 or

66.5% produced clouding in the antigen.

-9-

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Especial attention might be directed to lot A, where, of 179
sera all were clear except three which were roily. The roilies did not
produce clouding while 136 clear sera did. When after refrigeration for
one week, these sera were again examined no further roilies had appeared,
nor had any of the clear sera changed in character as far as could be
determined by visual inspection. After two weeks of refrigeration in-
spection of this lot showed seven additional roilies all of which had
originally clouded the antigenic suspension, and 4-2 precipitated sera,
which included two'sera which had given clean cut agglutination reac-
tions on the diagnostic test and lO hemclysed sera. This lot was then
placed at room temperature for 30 hours at which time the condition ‘
of the sera were again noted. Six additional samples showed precipita-
tion and of these, four had not clouded antigen, and the other two had
given clean out agglutination reactions, while one additional sample
was hemolysedo

It was noted in the course of this work that it frequently
became possible, when adding serum from precipitated serum to antigen,
to regulate the discharge of the sense fra the pipette so that no
visible particles were carried over. Moreover, in all transfers it
was the practice to wipe the tip of the pipette to remove serum from
its exterior and thus increase the accuracy of the measurement at trans-
fer; such cleaning probably aided in preventing the introduction of pre-_
cipitate into the antigen. Thus if the factor responsible for the forma-
tion of the cloudy reaction was completely segregated into the precipi-
tates contained in precipitated sera, the absence of the cloudy reac-
tion on such sera in a large number of cases might be anticipated,

specifically in those cases where the precautions just noted were can-

-11-

pletely effective in preventing precipitates from being carried over.
Since the procedure employed would however yield no certainty that small
quantities of precipitate had not in.faet'been introduced into the anti-
gen this cbservaticn can not be given its proper evaluation.

Conclusion: The condition of the serum obviously is no crite-
ricn which can be relied upon to predict whether a given serum'will pro-
duce a cloudy reaction in the agglutination system.

Data.were gathered on a limited number of sera to determine
the effect of hemolysis on clouding, and to learn whether clouding sera
which are hemolysed offer greater handicaps for accurate diagnostic
work than slight hemolysis or clouding alone.

In Table No. 4 the number and percentages of each type of
serum fra one shipment of blood samples from the same flock, and
handled under identical conditions are presented by a division into
those which showed laking in addition to various degrees of precipita-
tion and into those which were not hemolysed but showed varying degrees
of precipitation or were clear. An inspection of the table shows that
the percentages of cloudy reactions resulting from samples which were
hemolysed are uniformly higher than from.the non-hemolysed sera. It is
generally recognised that laked sera, and those giving the cloudy reac-
tion are not satisfactory for an accurate reading of agglutination. The
factors causing interference apparently tend to reinforce each other
when.both are present, and thus tend to intensify the interference each

would separately cause.

-12-

Table “Ge 4

Occurrence of Clouding in Serum as Influenced by Condition of Serum.

 

 

HEMOLIZED SERA NON-HEMOLIZED SERA

: No. Per cent : No. Per cent
__ : lumber Gloudi:ng Clouding: Number (Houdini Clouding
Clear 15 4 26.+ 100 11 11.0
Roily -- -- -- 5 6 100.0
Light Ppt. 28 9 32.0- 41 7 17.09
HOB” Ppte 22 15 68c. 6 2 33.3.
Totals 66 28 43.00- 162 25 16.40-

 

Relationship of Sex to the Warn“ of the Cloudxjeaction

In literature which has appeared heretofore one investigator
only, namely, Stafseth-G- has reported observations which relate to the
question whether sex of the bird has any relation to the appearance of
the cloudy reaction; this investigator did not encounter any blood samp-
les frmn male birds on which the cloudy reaction was obtained. vaiously
relatively few male birds are kept in breeding flocks and thus in a
season's testing, any observation which might be made on the point in
question would be based on far smaller numbers of birds than if some
factor which involved females were being studied. Two hundred and forty-
three male birds however became available for purposes of observation of
the occurrence of the cloudy reaction as against 2,981 females in a
total of 3,224 birds. Table No. 5p-esents the distribution of cloudy

reactions as among sexes.

~13-

Table NO. 5

A Comparison of Male and Female Birds giving Positive Agglutination
and Clouding Reactions

Flock Total: F E M A L E S 1 : M A L E S 1._
Key No.01}: fio.Re- TRe- No. 7 : N0.Re- ”5’ fie- N0. 7?
Letter Birds:No.actorsfiactors Cloudy Cloudy:No.actors actors Cloudy Cloudy

 

 

 

AH 403 374 38 10.1 11 2.9 29 0 0 0 0
an 324 290 33 11.3 84 28.9 34 1 2.9 2 5.9
c 86 84 6 7.1 15 17.8 2 o 0 0 0
E 373 323 58 17.9 56 15.0. 50 7 14.0 ' 10 -‘ 20.0
F 193 176 18 10.2 17 9.6 17 1 5.8 o 0
IP 464 441 1 0.2. 281 60.5 23 o 0 3 13.0.
J 181 172 26 15.1 5 2.9 9 0 0 0 0
K 100 95 10 10.4 28 29.4 5 0 0 0 ' o
L 99 94 3 3.2 13 13.8 5 1 20.0 o 0
u 133 115 10 8.7 18 15.6 18 1 5.5 0 0
u 519 490 24 4.9 84 17.1 29 1 3.4 0 _ o
o 349 327 22 6.7 82 23.5 22 o 0 0 0
Grand

762.1 3,224 2981 261 8.7. 694 21.5 243 12 4.9 15 6.1

A H,- Birds of groups A and H in T451. No. 6. These birds belong to the

same flock but were tested in separate groups. The designations
BD and IP are to be interpreted similarly.

Discussion: The records were gathered from tests which were

 

made prior to the breeding season. Thus at this time in many. of the
flocks, the. male birds were not kept with the females and on somewhat
different rations. The influence of a possible variation in the type
or quantity of feed thus prevents drawing conclusions on the influence
of the sex factor per se in leading to cloudy react ions.

That the cloudy reaction may be found however in tests on
serum from males is established, and it is further to be noted that
in one group, Flock E, 20% of the males reacted cloudy to only 15% of
the females. A number of flocks were found in which the serum of none
of the males produced the cloudy reaction which was contrary to what
might have been expected in view of the percentage of such reactions
encountered with serum from females in the same flock; note especially
Flocks A and O. The influence of feed above referred to may have been
accountable for this difference.

Conclusion: Sex is not the controlling influence in determin-
ing the incidence of the cloudy reaction.

Percentgge of Cloudy Reactions as Faun; in Individual Flocks; Rela-
tionship}; the Influence of Breeds on Clgdfl.

 

The statement has previously been made that other workers have
found as high as 75% of the birds of sane flocks give the cloudy reaction,
and that this reaction may cause such serious interference with diagnosis
by means of the agglutination test, that it has been recommended to con-
demn birds giving cloudy reactions (unless retested, in which event, the
possibilities are, according to Stafsetlgs- that such birds will continue
to give the cloudy non-specific reaction), as well as those giving the

positive specific reaction. Records gathered and presented here in Table

~15-

Table

N0. 6

Showing Incidence of Clouding and Cmparison of Cloudy and
Positive Reactions to the Agglutination Test.

Per Cent

Breed

 

Flock Number NuMber Per Cent Number
KEILL°ttQF Tested_Cloudyg Cloudy Reactors Reactors
A 182 3 1.69 25 13.79
B 101 36. 15.69 12 11.99
C 86 15 18.49 6 6.99
D 223 50 22.49 22 9.89
E 373 66 17.69 65 17.49
F 193 17 8.89 19 9.89
G 180 136 75.69 16 8.89
H 221 8 3.69 13 5.89
I 317 174 54.89 0 0.0
J 181 5 2.79 26 14.39
K 100 28 28.05 10 10.0
L 99 13 13.19 4 4.09
M 133 18 13.59 11 8.29
N 519 84 16.19 25 4.89
0 349 82 23.49 22 6.39
P 147 110 74.89 1 0.69
‘Tctal
Light Breeds 1697 614 36.19 74 4.39
Total
Heavy Breeds 1707 231 13.59 203 11.39
Grand
Total 3404 845 24.89 277 8.19

‘Wh. Plymouth Rock
'Wh.'Wyandctte

Wh. Leghorn
'Wh.'Wyandctte
Barred Rock
Barred Rock

'Wh. Leghorn

Wh. Plymouth Rock
lh. Leghorn
Barred Rock
'Ih.‘Wyandotte
Brown Leghorn
Barred Rock

‘Wh. Leghorn

‘Wh. Leghomn

Wh. Leghorn

 

~16-

No. 6 are of interest in this connection. Records which show the in-
oidence of the cloudy reaction as between the sera of different breeds
are also presented in the table. Both sexes are included.

Discussion: The highest percentage of clouding encountered

 

was 75.5% which corresponds with the values reported by Hathews. The
lowest percentage encountered was 1.6%, and the average for all birds
of the 16 groups was 24.8%.. It would appear then that so drastic a

recommendation as advising the condemnation of birds giving a‘ cloudy
reaction might in many instances cause a severe hardship for certain
flock owners, and undoubtedly would alienate mam poultrymen from

support of a system of control of bacillary white diarrhea based upon

the elimination of carriers of S. pullorum infection as indicated by the

 

agglutination test.

The success of any plan for the control of bacillary white
diarrhea by elimination of infected adults depends of course upon the
accuracy of the diagnostic procedure, or rather its reliability in not
leaving infected birds in the flock. It depends also however upon the
ability of the plan to secure and maintain popularity with the flock
owner by not causing excessive inconvenience or inflicting excessive
losses.

The foregoing table presents records on a few breeds only.
Blood samples frm birds of breeds which are not represented, were not
obtained. While 36.1% of samples fran birds of the light breeds and
13.5% of birds of the heavy breeds gave the cloudy reaction, no clear
out differences as between breeds can be made, for Flock L of Brown Leg-
horns yielded 13.1% and Flock D of White Wyandottes yielded 22.4% cloudy

reactions . .

-17-

Conclusions; The incidence of the cloudy reaction is quite
variable as between blood samples of different flocks. In some in-
stances it may rarely occur (or be absent) and in others be as high as
75.5%.

The breed of birds was not found clearly to influence the in-
cidence of the cloudy reaction, although on the whole, light breeds in

the work here presented yielded higher percentages of cloudy reactions.

-18-

SECT ICN II

CHEMCAL NATURE 03‘ THE CLOUDING FACTOR

Warner and Edmond-.3- showed that there is a higher percentage

of fat in the whole blood of pullets and hens in heavy lay than in
females not in lay and in male birds.‘ Reasoning from this basis
Hitchneri conducted a number of tests to determine whether fasting
before bleeding tended to reduce the percentage of sera showing cloud-
ing and found that this practice had the anticipated effect. He per-
formed no chemical tests but reasoning from the basis of the work of
Warner and Edmond stated that fat contained in the serum was the factor
responsible for cloudy reactions. That fasting does reduce the number
of cloudy reactions has been amply confirmediz

Mathews-l‘ conducted chemical tests for protein on the precipi-
tate obtained from two lots of chicken serum. He secured a figure of
147! nitrogen and a confirmatory test for protein by the biuret and
xanthoproteic test on the one serum and by the biuret test only on the
other. He then concluded that protein was the factor responsible for
clouding.

Further reference to Mathews' work shall be made after the
data on the chemical tests here undertaken have been presented. These
tests included ultramicrosoopio observation for the presence of fat in
sera, micrcchemical tests for a qualitative determination on the pres-
ence of fats, and quantitative tests using a Soxhlet extractor for the
percentage of fats occurring in the precipitates formed when clouding

sera were added to standard antigen.

~19-

Ultramicrosccmic Observations of Sara

Following the method for the ultramicrcscopic demonstration
of fat globules in the blood introduced by Neumann'fe' and Oshimag, the
following kinds of sera were observed by means of the ultramicroscopc:
(l) precipitated sera, and (2) clear or non-precipitated sera, which pro-
duced clouding in the agglutination system; and (3) normal, i.e., clear
non-clouding sera. The sera were subjected to centrifugation at 2,000
r.p.ll. for one hour. The precipitated sera prior to centrifugation appear-
ed milw or somewhat curded; after centrifugation a layer of material of
a cream to light yellow color was observed at the top of tin tubes. This
layer was of variable thickness and ranged in volume from 0.08 to 0.2
mils , from an original volume of 2 mils of blood. In the clear sera
which produced clouding, a delicate film of material was observed at the
top of the tubes. In the normal sera, it was also possible to see a
film in a few cases.)

Portions of the precipitate were resuspended by trituration in
distilled. water to gain a suitable preparation for microscopic examina-
tion. Where delicate surface films only were yielded by centrifugation
these were sampled for examination by means of the platinum loop. A 6
ampere arc was used as the illuminant with the ultra condenser of the
ultramicrcs cope .

Fat globules were revealed in all cases. Sera yielding heavy
layers of precipitate upon centrifugation showed amorphous masses as well.
The globules were larger and more numerous in the suspensions from the
precipitated sera, than in the preparations from: normal sera, where the
globules were observed more infrequently, and to be of much smaller size.
The supernatant film from sera which when not diluted did not precipitate

but which when added to antigen produced the cloudy reaction appeared to

have a lower fat content than the precipitates.

Additions of 0.02 and 0.01 mils of the suspended precipitates
to 1 mil of standard antigen, corresponding to the regular 1-50 and 1-100
dilutions in the routine agglutination test, invariably produced clouding.

These relationships were regularly encountered in a representa-

tive number of sera of each of the types studied.

Microchemical Test:

Samples of the precipitating material which remained in suspen-
sion or floating on the surface of the fluid in the serological tubes
showing clouding on the agglutination test were secured. This material
was obtained either by dipping with a platinum loop and placing it on
a slide for microscopic examination, or by allowing the liquid to dry
down in the tubes in a place free from vibration in which case the pre-
cipitating material would adhere to the sides of the tubes; the dried
or semi-dried material was then recovered by scraping.

The dried material was suspended in a drop of distilled water
and triturated by means of a platinum loop, covered with a cover-slip,
and then examined under the microscope with the 4 m.m. and oil immersion
objectives. Amorphous and globular floccules were observed. With the
field under observation Sudan III was introduced under the cover-slip by
drawing a drop of the stain into the preparation by means of moistened
filter paper. Examination now showed definitely stained globules like
then characteristic of typical neutral fats, and other angularly ovoid
approaching the configurations which are recognized to be presented by
fatty acids .32

Alsc long slender needles were observed the shape and staining
qualities of which were characteristic for fatty acid crystals. Other

material of an amorphous structure were observed in the preparation.

Other similar preparations were treated with 36% acetic acid,
covered with a cover slip, heated until bubbles arose and then examined
while cooling under the microscope. This methcd'was successful in some

1
cases in revealing fatty acid crystals.-g

Ether Extraction by_the Scxhlet Method

lethcd of Procedure: Four sera in which no precipitation

 

occurred in the undiluted state but which showed varying degrees of cloud-
ing 0 n the agglutination test were used to determine what percentage of
the precipitate was soluble in ether. This is the standard method for
extraction of fat and cholesterol, used by Warner and Edmond in their
determination of the blood fat in the sera of domestic fowls. T0 19 0.0.
quantities of standard antigen in.test‘ tubes, l 0.0. of each of the sera
were added, and the mixtures were incubated for 24 hours. Again varying
degrees of clouding were noted, which ranged from.a diffuse turbidity, to
suspended floccules, to a heavy floating suspension of floccules, and to
sedimentation of the floccules. The liquids were filtered through tared
filter paper, and the filter papers dried in an oven at 37° for 24 hours.
The filters with the dried precipitate were again weighed and then placed
in the thimbles for extraction. Extraction was conducted for 16 hours.
The percentages of ether soluble matter secured from.the dried
flocculating precipitate were 7.3, 8.2, 11.2, and 31.1%. See Table No. 7.
These percentages bore a direct relationship to the degree of clouding
noted in the several tubes prior to filtration. The high value of 31.1%
was secured from a serum in which considerable clearing in turbidity of

the antigenic fluid, besides large floccules, occurred.

-22...

Table N0. 7
Ether Soluble Matter Contained in Precipitates Resulting from
Addition of Clouding Sera to Standard Antigen

 

Serum Wt. Dried Wt. Ether 7'3 Ether Degree of

Number Rpt. Gms. Extract Gms. Extract Clouding

291 0.01345 0.00110 8.2 Turbid

296 0.01335 0.0015 11.2 Turbid

344 0.0108 0.00315 31.1 Large Floccules with
clearing

352 0.0034 0.00025 7.5 Slightly turbid

Discussion: In discussing the results of this work it appears

 

desirable to refer to the work of biochemists which bears on the question
of fats in the blood plasma. Neisser and Braeuningll'and other workerslg
have shown by work on mammalian serum: (1) that serum is almost always
milky shortly after the ingestion of fats; (2) that in extreme cases
fat may on standing separate as a creamy layer from the serum; (3) that
ultramicroscopic particles of fat can be demonstrated; (4) that the quanti-
ties of fat in the blood, determined by other extraction, vary with the
amount of fat ingested; and (5) that the above criteria and measures of
fat in the serum are absent or reduced following a period of starvation.
Several analogous findings on avian blood serum with respect to its fat
content are here presented. The fact that workers have found the prac-
tice of starving fowls a period of 48 hours a means of satisfactorily
minimizing the clouding reaction is in harmony with the last cited Obser-
vation that starvation reduces the fat content of a serum.

The investigations here undertaken on precipitated and clear sera

which produced clouding in the agglutination system, and on clear non-

-931-

clouding sera, in which ultramicroscopic studies on the presence of fat
were made, showed that a correlation may exist between the fat content
of a serum and its tendency to produce the cloudy reaction. The micro-
chemical tests on the precipitates found in standard antigen when sera
which yielded the cloudy reaction were added, showed an abundance of
fats and fatty acids present. This lends weight to the view that fat
is involved in the appearance of the cloudy reaction.

The quantitative data on the amount of fat in precipitates formed
in standard antigen when precipitating sera were added, which was somewhat
variable but in the sera investigated never lower than 7%, must be re-
garded as secondary in conclusiveness.

The precipitate must contain (1) the substance which is in such
unstable condition in the agglutination system that it tends to precipi-
tate out, (2) bodies of bacteria carried down in the precipitate by ad-
hesion or occlusion, and (3) blood serum and bacterial colloids present
in the system carried down by occlusion and also by adsorption to the
precipitating substances. Thus, it may be seen that a very small quan-
tity of material in an unstable equilibrium in the agglutination system,
whether it be lipoidal or protein in nature, might cause the precipita-
tion of several times its mass of other materials; and thus the quanti—
tative tests did not show the amount of fat, if any, which was responsi-
ble for initiating the precipitation.

The method used by Mathews for securing precipitates for quanti-
tative analysis on the basis of which he concluded that protein was res-
ponsible for clouding should be noted. FromKMathews' paper it appears
that he took precipitates which formed in the‘whole serum of birds whereas
in the work here reported precipitates which formed in a standard antigen

were employed. In the former case the percentage of protein (and other

-24-

substances) mechanically carried down during precipitation should be
anticipated to be much higher than in the latter where a\l-20 dilution
of serum was used. Blood plasma is largely a solution of albumins and
globulins which are colloids and which according to the principles of
physical chemistry should be expected to be carried out of solution in
the presence of a precipitation even though that precipitationhhriginr
ated in or primarily concerned some other constituent of the plasma.

It is not surpising then that Mathews should have found the
precipitates which he analysed to contain 14%:nitrcgen, but this does
not prove that protein caused the precipitation.

We must conclude that quantitative examination of precipitates
as formed in blood serum, or as formed in standard antigen does not give
direct evidence as to the nature of the substance which caused the pre-
cipitation. Yet of the two methods, the examination of precipitates
formed in standard antigen may be considered to be the more reliable
since such antigen probably contains less mass of materials which might

be carried accidentally than does blood serum.

Conclusions: Examination of precipitated sera, non-precipitated

 

clouding sera, and nonmal, non-clouding sera of chickens by means of the
ultramicrcscope revealed fat globules in all cases. The fat globules are
present in greater quantities in precipitated and in non-precipitated
clouding serum than in non-clouding serum.

Microchemical tests on and quantitative fat determinations of
the precipitate resulting in the agglutination system from the addition
of clouding sera, show that precipitate to contain large quantities of
neutral fats and fatty acids. These facts are highly suggestive that

fats are responsible for the cloudy reactions.

 

ponsit 10 f
in the 11g
the facts

as discus:

Evidence that has heretofore been adduced that protein is res-
ponsible for the clouding phenomenon does not appear to be well founded
in the light of the experimental results here presented, and in view of
the facts established by scientists on related phenomena in other species

as discussed in this section.

SECT ION III

SOURCE CF THE PRECIPITATING mATERIAL

It was thought desirable to determine the source of the cloud-
ing factor, i.e., to learn whether elements contained in the blood serum
or contained in.the bacterial suspension furnished the preponderance of
the clouding substance, in order that a logical attack on the problem
of finding a means of inhibiting the reaction might be had.

Method of Procedure:
A series of cross-agglutination tests was run on ten clouding

sera. The antigens used were mixtures of five strains of Salmonella

 

pullorum suspended in 0.5%Lphenolised saline and having densities of 0.25,
0.5, 1.0, 2.0, 3.0 and 5.0 according to the McFarland Nephelometer scale.
Seven dilutions of each serum ranging from.l-20 to 1-1280 were used
against each of these antigens and also against physiologic saline con-
taining 0.5%.phenol. Observations on the appearance of clouding were
made at the end of 24 and 48 hours after setting up the test.

It immediately became apparent that some standard must be estab-
lished in order to make the observations objective and canparable. A
comparator was devised by the use of which it was possible to assign
values in terms of turbidity on the MdFarland nephelometer scale to de-
grees of resultant turbidity occurring in each tube in the test.
Description of the Comparator:

A set of tubes of the various densities of antigen was used as
the standards. The tubes were of the same dimensions, i.e., 8 m.m. by
75 m.m., as those used in the test, and these were mounted in a rack‘which
had provision for placing the tube to be read in the row parallel with the

standards. On the lower cross-member of the rack, and immediately back

of the row of standards was placed a strip of white bristol board upon
which there was ruled a fine line with India ink. A 40 watt Mazda lamp
with a reflector was used as the light source and so arranged on the labo-
ratory table that a neutral background was obtained. By holding the compar-
ator parallel with the illuminant, it was found that by holding it upright,
or by tilting it so that a greater depth of liquid was interposed between
the black line and the eye of the observer, objective readings of the de-
grees of turbidity could be made.

The readings were recorded by an assistant so as to remove any
influence which might arise from.noting previously recorded readings. As
a further check another worker was asked to read several of the series of
tests; the two readings were found to agree closely; no variation being
greater than a plus or minus at any degree in the scale.

‘Where flocculation or sedimentation had occurred the sane'was
recorded and the tubes were then agitated to break up the aggregated ma-
terial in order to get a true reading of the precipitation encountered.

Control tubes to which no serum had been added were run on all.
antigens. It should be noted that in controls sedimentation of the bacterial
suspension occurred in the heavier densities of antigen, particularly in
the No. 5 and 3, and to some extent in No. 2. But since the control tubes,
in no case upon thorough agitation yielded a turbidity above that of the
original density of the antigen, they were omitted from the tables. They
served as check upon the sedimentation due to natural settling and upon
the increase in turbidity due to the clouding factor, as well as upon tur-
bidity which might have resulted from bacterial growth or other causes.

Tabulated results on the ten sera are presented in Tables 8 to
17 inclusive. Under the heading I'States of Clouding”, the terms used are

to be interpreted as follows:

-23-

Table No. 8

Influence of Density of Antigen on Appearance of Clauding.
Serum No. 289. Final, i.e., 48 hr. readings presented.

DENSITY OF ANTIGENS

 

 

Phenol
Serum States of Saline
Dilutions Claudingg'Control 0.25 0.5 1.0 2.0 3.0 5.0
Suspended - - - - - - a
Sedimented - - - - - - -
1-20 Diffuse e e e e e a e
Incr. Turb. 4 4 3 3 2 2 e
Suspended - - - - - - -
1-40 Diffuse e e e a e e e
Incr. Turb. 4 4 - 2 e 2 l l Tr
Suspended e - - - - - -
Sedimented e - - - - - -
l-80 Diffuse - e e e e e -
Incr. Tuwb. 3 3 2 l l - Tr -
Suspended e - - - - -
Sadmgnth ’ - e- e- o - e-
1-160 Diffuse - e e e a - -
Incr. Turb. 2 2 l Tr Tr - -
Suspended a e - - - - -
Sedimented e e - - - - -
1-320 Diffuse - - 9 - - - -
In". Turb. 1 1 Tr - - -
Suspended e e - -
Sedimented e e - - - -
1-640 Diffuse - - - - - - -
Inore Turb. 0e5 0e5 - - - — -
Suspended - - - - - - -
Sedimented e a - - - - -
1-1280 DEfU.‘ . - - :- ce .- o
lncr. Turb. Tr Tr - - - - -

Table “Ge 9

Influence of Density cf Antigen on Appearance of Clouding.

Serum No. 344.

DENSITY (F ANTIGENS

Final, i.e., 48 hr. Readings Presented.

 

 

* Flcccules floating on surface.
** Floccules very minute.

Phenolized
Serum ' States of Saline
Dilutions Glouding, Control 0.25 0.5 1.0 2.0 3.0 5.0
Suspended e s as as e - - -
1-20 Diffuse - - - + g ,
Incr. Turb. 5 e 5 5 e 3 2 Tr.
Suspended 9* as . ass - - -
1'40 Diffuse O u- - .- ¥ 1- .-
Incr. Turb. 5 4 3 4 2 l
Suspended e e e sea - - -
Sedimented - - - - - - -
. 1-80 Diffuse - - - e e e -
Inor . Turb . 2e 2 2- 2 l Tr -
Suspended e e e as! - -
Sedimented - - - - - - -
l-lGO Diffuse e - e e 9 - -
Inor. Turb. 2 19 1 1 Tr ' ‘
Suspended + e s set -
Sedimented . '- v as — us .—
1-320 Diffuse e - e e - - -
nor 0 Turb e 1" 1. TI‘ TI‘ - I- -
SUBpOndOd " - II I- as as -
SedimentOd - - fl - u e- -
- 1-640 Diffuse r e - - - - -
Incr. Turb. C 5 0.5 - - a u -
Suspended - - - - - -
Sedimented - C u D '- . .
l-1280 Diffuse e p - - - - -
Control - - — - - -

 

 

 

 

 

 

I I O .
r _ _ _ .. .. _ w _ _ .n. _ i
_. _. . .
_ _ _ .. _ _ —. . _ . _ u _ ..
T e
. a .. . _. _ _. _ _ _ _ . a _ _
— . .
_ _ . , u. n r _ _ .. . _ _ _ _ _
H _ _ 1 1 .. _ _ — _ A _ . _ _ a a
, _ _ . s s . _ _ _ _ _ _ _ _ _ .

 

 

Table No. 10

Influence of Density of Antigen on Appearance of Clouding.

Serum No. 340.

Serum

Dilutions

1-20

1-40

1-80

1-160

14320

l-640

1-1280

States of
Clauding

Suspended
Sedimented
Diffuse
Incr . Turb .

Suspended
Sedimented
Diffuse
Incr. Turb.

Suspended
Sedimented
Diffuse
Incr. Turb.

Suspended
Sedimented
Diffuse
Incr . Turb .

Suspended
Sedimented
Diffuse
Incr. Turb.

Suspended
Sedimented
Diffuse
Incr . Turb .

Suspended
Sedimented
Diffuse
Incr. Turb.

Phenol
Saline
Control

NQII

HQII H'PI

H9

DENSITY CF ANT IGENS_

0.25

N‘l’ll

-31-

N?ll

Final, i.e., 48 hr. readings presented.

H? I 10""

Illl I IH'PII

IIII

Table No. ll

Influence‘of Density of Antigen on Appearance of Clouding.

Sernum NOe 296e

Serum

‘Dilutions

1-20

1-40

1-80

l-l60

1-320

1-640

1-1280

States of
Clouding

Suspended
Sedimented
Diffuse
Incr. Turb.

Suspended
Sedimented
Diffuse
Incr. Turbo

Suspended
sedimented
Diffuse
Incr. Turb.

Suspended
Sedimented
Diffuse
Incr. Turb.

Suspended
Sedimented
Diffuse
Incr e Turb e

Suspended
Sedimented
Diffuse
Incr. Turb.

Suspended
Sedimented
Diffuse
Incr. Turb.

Phenol
Saline
Control

DENSITY CF ANTIGENS

0.25

HI'OI NI‘II

‘9

0.5

I 2.".

f

N
.'

I-‘I'PI

1.0

HI?! NI‘I’I OII‘I'I OFI'PI

Tr

Final, i.e., 48 hr. readings presented.

2.0

'0

HIQI NI’I OIIQI .1:

1

FBI
e.

3.0

‘I'

2+

‘I

NIQI I

I-‘IQ’I

5.0

QI'I'I

{IQI

 

 

. O I
a _ _ _ . . _ _ g _ _ _ _ _ _
a _. _ _ A A _ . _.. . e _ .T .

*

.H.
— V . . _. . _ T . e — -
_ _ n , A _ ... _ —. _
. ~. _ _ _. _ . ._. . _
. _ ~ ~ _ . _ a . . _

u.

. —. _ 7 I _. - H _ _ _ _ _ a

   

la: 4".
a . I..

 

 

Table No. 12

Influence of Density of Antigen on Appearance of Clouding.

Serum No. 298.

Final, i.e., 48 hr. readings presented.

DENSITY OF ANTIGENS

 

 

Phenol
Serum States of Saline

‘Dilutions Cloudigg Control 0.25 0.5 . 1.0 2.0 3.0 5.0
Suspended e - - - - - -

Sedimented - e e e e + +

1-20 Diffuse - - - - - - -
Incr. Turb. 0.25 1+ 4 4 4 2+ e

Suspended - - - - - -

Sedimented - e e e e e e

1-40 Diffuse e - - - - - -
Incr. Turb. 0.25 l 3- 3 3e 2e 6

Suspended - - - - - - -

Sedimented - e e e e e +

1-80 Diffuse e - - - - . - -
Incr. Turb. Oe5 1" 2 2 3 2* Tr

Suspended - - - - - - -

Sedimented - e e e e e -

l-160 Diffuse e - - - - - -
Incr. Turb. l- 0.5 l l 2 2 -

Suspended e - - - - - -

Sedimented - » e e e e e -

1-320 Diffuse - - - - - ~ -
Incr. Turb. 1 OeB- Tr Tr 1 1 '-

Suspended e - e— - - - -
.Sedimented ~ e - - - - -

1-640 Diffuse - - - - - — -
Incr. Turb. 0.25 Tr - - - - -

Suspended e - - - - - -

Sedimented - - - - - - -

1-1280 Diffuse - - - - ~ -
Incr. Turb. Tr - - - - - -

-33-

 

. .
. .
_ _ .. _ _ _. . _
_. _ . n _. _ _
. . . ..e _ _
_
. _ _ . . A
n . _ . . _. .
r _ _. m
. _.
_ _ .. _ _. . . .

 

I .Ie.r,h.lvr«llp.5
.... .V . 1

“It. .. ‘ ..

. . :1.
”in...“ . ”.1!!!

 

 

Table No. 13

Influence of Density of Antigen on Appearance of Clouding.

Serum No. 291.

DENSITY CF ANTIGENS

Final, i.e., 48 hr. readings presented.

 

 

Phenol
Serum States of Saline

Dillltion' Cloudigg_ Control 0e25 Gas 1.0 ZeO 3.0 5.0
Suspended + - - - - - -

Sedimented + + + + + +- +

l-20 Diffuse - - - - - - -
Incr. Turb. 3 2 2+ 4 3+ 2+ +

Suspended e - - - - - -

Sedimented + + + + + + +

1-40 Diffuse - - - - - - -
Incr. Turb. 2+ 1 2- 2- 3 2+ +

Suspended + - - — - - -

Sedimented - + 'e + + + +

1—80 Diffuse - - - - - — -
Incr. Turb. 2 1- 1+ 1+ 2 2 -

Suspended + - - - - - -

Sedimented - + + + + + +

l-160 Diffuse - - - - - - -
Incr. Turbo 1’ T? Gas 1 1 1 TI‘

Suspended - - - - - - -
Sedimented + - + + + + -

l-320 Diffuse - - - - - - -
Incr. Turb. l - Tr 1+ 1 Tr -

Suspended - - - - - - -

Sedimented + - - + + - -

1-640 Diffuse - - - - - - -
Incr e Turb e 0 e 5 - ~ 1 Tr . - -

Suspended . - '- - e- - .-

Sedimented + a - + - - -

1-1280 Diffuse - - - - - - -
Incr. Turb. Tr - - Tr - - -

Table NOe 14

Influence of Density of Antigen on Appearance of Clouding.
Serum No. 352. Final, i.e., 48 hr. readings presented.

:Serun

IJilutions

1-20

1-40

1-80

l-lGO

1-320

1-640

1-1280

DENSITY OF ANTIGENS

Phenol
States of Saline
Clouding Control 0.25 0.5 1.0

Suspended
Sedimented
Diffuse
Incr. Turb.

Suspended
Sedimented
Diffuse
Incr e Turb e

II
0-39
"I
0
e?
H?

I

Suspended
Sedimented
Diffuse

Incr e Turb e

I 1
9

Suspended
Sedimented
Diffuse
Incr. Turb.

Suspended
Sedimented
Diffuse
Incr. Turb.

Suspended - -
Sedimented
Diffuse
Incr. Turb. ‘

I
'I

Suspended
Sedimented
Diffuse
Incr. Turb.

-35-

2.0

3.0

*

HI‘II

9

Tr

5.0

QIOI

_ . .
. ,0 .e
_ _ _ _ . n _ _
. . . _ . .., _
_ _ . n i _. . _
.
_ .. . _ .. l

. _ . _ m n
_ .:

i. a . : , _ .

_. . a. _ _. _ ‘

 

. J. .. 1...: 5,!3.
r.
.3

  

 

Tfibl’ “Ge 15

Influence of Density of Antigen on Appearance of Clouding

Serum NOe 292e

Final, i.e., 48 hr. reading presented

Anansrrr or ANTIGENS

 

 

Incr. Turb. Tr

- Phenol
Serum States of Saline

IJilutions Clouding; Control 0.25 0.5 1.0 2.0 3.0 5.0

Suspended - - - - - - -

Sedimented + + + + + + +

l-20 Diffuse - - - - - - -

Incr. Turb. 0 .25+ 2 3- 3 3+ 2+ +

Suspended - - -‘ - - - -

Sedimented + + + + + + +

1-40 Diffuse - - - - - - -

Incr. Turb. 0.25 l 2 2 3 2+ +

Suspended - - - - - - -

Sedimented + + + + + + +

1-80 Diffuse - C C o a u -
Incr. Turb. Oe25 0e“ 1. 1" .2 2 Tr

Suspended '- I- c- a- - a- .-

- Sedimented + + + +' + + -

l-lGO Diffuse I- In - - c- - .

Incr. Turb. 0.25 0.5 l Tr 1+ 1 -

Suspended - - - - - - -

Sedimented + + + + + + -

l-320 Diffuse - - - - - - -

Incr. Turb. 0.25— 0.25 Tr Tr 1 Tr -

Suspended - - - - - - -

Sedimented e + - - - - -

1-640 Diffuao C D - - o u -

1110?. Turbo 0e25- Tr '- I- — u- -

Suspended - - e- n- .- - -

Badmntod .' G C D we so -

‘ 1-1280 Diffuse - - - - - - -

 

~36-

TEDIO No. 16

Influence of Density of Antigen on Appearance of Clouding.
Serum No. 324. Final, i.e., 48 hr. readings presented.

DENSITY CF ANTIGENS

 

Phenol
Serum States of Saline
Dilutions Cloud_ing; Control‘ 0.25 0.5 1.0 2.0 3.0 5.0

Suspended -

- - 9 - - -

Sedimented - - + + + + +

1-20 Diffuse e + - - - - -
Incr. Turb e 0 e 5 1 2 3 2 2 f

Suspended - - - + - - -

Sed imented — - - e- e- e a.

1-40 DiffuBO .- e- e - - - ..
Incr. Turb. 0.25 1- 1+ 2 l l Tr

Suspended - - - - - - -
Sedimented - - + + + - -

1-80 Diffuse a q- a - .- .. .-
Incr. Turb. 0.25 0.5 l- 2 Tr - -

Suspended - - - - - - -

Sedimented - - - + a -

1-160 Diffuse + + + - - — -
Incr. Turb. Tr Tr Tr l - - -

Suspended e - — - - - -

Sedimnted - s. O In - u -

l-320 Diffuse + - - - - - -
‘ Incr. Turb. Tr - - - - - -

Suspended - - — - - -

Sedimented - - - - - - -

1-640 DufuCO II '- wI - u. — -
Incr. Turb. - - - - - a -

Suspended - - - - - i -
SedmntOd - - d e- I- n .-

1’1280 Diffus. III e- n - — - .-
Incr. Turb. '- - es .- - a -.

Influence of Density of Antigen on Appearance of Clouding.

Serum No. 287. Final, i.e., 48 hr. readings presented.

Table No. 17

DENSITY CF ANTIGENS

 

 

Phenolized
Serum States of Saline

Dilutions CloudinL CODtI‘Ol Oe25 0.5 1.0 2 e0 3.0 5e0
Suspended + - - - - - -

Sedimented - —v - - - - -

1-20 Diffuse + + + + + + —
Incr. Turb. 1+ 1 l. l l l -

Suspended + - - - - - -

1-40 Diffuse + + + + + - -
Incr. Turb. l l 0.5 Tr Tr - -

Suspended - - - - - - -

Sedimented - - - - - - -

1-80 Diffuse + a a - - - -
Suspended - - — - - - -

Sedimented - - - - - - -

1-160 Diffuse + + - - - - -
Incr. Turb. 0.5+ l- - - - - -

Suspended + - - _ - - -

Sedimented - - - - - -

Incr. Turb. 0.5 0.5+ — - - - -

Suspended + - - - -

Sedimented e- f .- - .. -

1-640 DifoBO " '- I- .- - u -
Incr. Turb. .25 Tr .-. - .- - ..

Suspended + - - — - - -

1-1280 Incr. Turb. Tr - - - - - -
<3ontrol - - - - - - -

-38—

Suspended: The precipitate appears as floccules which are in
suspension at the surface or throughout the medium.
SedimentedzThe precipitate has settled out at the bottom.of
the tube.

Diffuse: The precipitate is in a finely divided state, and
evenly dispersed throughout the medium.

Increased Turbidity: The *nume'nsls indicate the resultant in-
crease in turbidity over that of the original density of the
antigen according to the McFarland nephelometer scale.

 

 

Discussion:

 

An examination of the tabulated results on the various sera
reveals slight variations in details but a well marked general trend.
It was found that heavy precipitates may appear when clouding sera are
added to phenolised saline and that in such mixtures the amount of serum
present bears a definite relation to the quantity of precipitate. More-
over, as the density of the antigen increases less precipitate is formed
at a constant dilution of serum; and in the heavier densities of antigen
no precipitation or clouding was detected in the more dilute serum. This
may also be stated thus: that the heaviest precipitates and the appear-
ance of precipitates in high dilutions of serum occurred where pheno-
lised saline served as the serun diluent or where antigen of low density
served in that capacity. It appears, therefore, that a heavy suspension
of antigen tends to inhibit clouding. This behavior may be referable
to the adsorption of the unstable element, the precipitation of which is
responsible for the appearance of clouding, to the bodies of bacteria in
the test fluid, in consequence of which this element becomes inoperative.

An inspection of the tables shows further that the precipitating
Inaterial may manifest itself by floating at the surface, or by being in
suspension throughout the medium, or by forming a flocculent sediment
at the bottom of the tube, or by being in a.minutely divided and evenly

dispersed state throughout the medium, or by appearing as a combination

«b

of two or more of these states; and that these states are all manifesta-
tions of the same phenomenon differing only in their degree of aggrega-
tion or dispersion. A floating precipitate will commonly settle out
when the equilibrium is disturbed as by jarring. The precipitate may
begin to settle down in the form of long strands or as floccules of
variable size. A diffuse clouding is due to a dispersion of the minute
particles of the precipitate in the test fluid.

Conclusions: The question regarding the source of the major

 

proportion of the precipitated material, whether from serum or from
antigen was not fully answered. 'Yet the results on mixtures of serum
" and phenolised saline where in the absence of antigen heavy precipitates
occurred indicate that the serum probably is the principal source of the
precipitate.

Definite evidence was obtained indicating that a heavy suspen-

sion of antigen in itself tends to inhibit the cloudy reaction.

SECTION IV

DWESTIGATIQI ON MEANS a" PREVENTING THE PRECIPITATIGSI
0F SUBSTANCES RESPONSIBLE FOR CLOUDING

Early in the course of the study, two possible means of
escape from the clouding reaction suggested themselves. These were,
the adjustment of the temperature and holding period of the agglutin-
ation system, and the observation of a minimum of disturbance to
the tissues at the point of incision in drawing the blood samples.

Manipulation of the AgglutinationASystem by Alteringgthe
Temperature

 

 

Reasoning from the basis of Madsen'slg’work which showed
that the rate of agglutination reaction increases with the tempera-
ture, as long as this is not high enough to injure the reacting sub-
'stanees, it seemed possible that a temperature and holding period
might be found which would be selective, i.e., favor the agglutina-
tion reaction and inhibit the cloudy reaction. The tests undertaken
were as follows; 37 sera which clouded on the routine test were run
simultaneously on standard antigen which was incubated continuously
at 37° C for 24 hours, followed by refrigeration for 24 hours, on
standard antigen incubated at 60° 0 for 4 hours, followed by 15 hours'
refrigeration; on non-phenolised antigen incubated at 37° C for 6
hours and refrigerated for 12 hours; and on non-phenolised antigen
incubated for 6 hours at 50° 0 for 6 hours followed by refrigeration
for 12 hours.

The results were negative; clouding occurred uniformly in

each of the series subjected to various periods of incubation and

-41-

l‘

refrigeration, though differences in the degree of clouding were noted

in the reactions produced by individual sera in the four methods tried.

Influence of the Method of Drawing Blood Samples

 

The method of nicking the ulnar or humeral veins for securing
blood samples of chickens in field work is usually accompanied by a
minimum of aseptic precaution, and the method does not always yield a
free flow of blood. Ooservation of the operations of bleeders showed
that it is a common practice where a poor flow of blood results after
nioking, to pump the wing of the bird and to irritate the wound by scrap-
ing with the lanes or lip of the tube until a sufficient sample is secured.
It was thought that possibly such irritation which is occasionally pro-
longed causes enough scurf and epithelial cells to be lessened from the
skin, and enough destruction of the exposed tissues so that the quantity
of cellular elements and debris entering the blood sample was sufficient
to influence the behavior of the serum and be a factor in the production
of clouding.

Twelve birds were bled to determine this point. Blood samples
were secured from both wings of each bird. In taking the first sample
care was taken to secure a clean out which produced a free flow of blood
without any further irritation. The second sample from the other wing was
taken by nicking and then subjecting the wound to excessive scraping and
irritation. None of the duplicate samples thus secured produced cloud-
ing. Further evidence that mechanical injury to the wound in bleeding
is not responsible for clouding was secured on a pen of 84 birds. Approxi-
mately half of this flock was bled by an inexperienced bleeder who resorted
to irritation to secure adequate samples, while the other half of the flock

was bled by the writer who took precautions to insure a free flow with a

42-

single incision and without irritation. The two lots of samples secured
showed no notable differences in the percentages of cloudy reactions on
test; the percentage of cloudies on the whole flock'was 76. The method
of bleeding is thus shown to have a negligible influence on the appearance
of clouding.

Phenol Omitted from Antigen for Avoidance ofglouding

 

Rebraisserli reported in a series of studies that the omission
of phenol from antigen was followed by the disappearance of interference
from the clouding reaction. If this were generally true it would offer
a ready means of solving the problem. In order to make a comparison of
the appearance of the clouding reaction on phenolised and non-phenolised
antigens three groups of non-agglutinating sera totaling 266 samples were
used in duplicate on such antigens. The serum-dilution employed in each
case was that of l to 60; the antigen of a density of tube No. l nefarland
nephelometer and containing 0.85%‘NaCl; the incubation at 37°. Final or
48 hour readings are presented in Table No. 18 which shows the results.

Table No. 18

Comparison of Occurrence of Clouding Reaction in Three Groups of Sara
when used against Antigens Prepared with and without Phenol

 

 

 

: : : :Unchanged:Unchanged
Number: ANTIGEN A t : ANTIGEN B as :Unchanged: on A : on B
0! z : 3 z : on : Clouded : Clouded
Sera :Clouded:Unchanged:CloudedzUnchanged: Both : on B : on A
146 110 36 145 1 1 35 0
173 73 100 78 95 85 12 8
37 19 18 14 10 10 9 12
Totals
256 202 164 237 119 96 56 20

* Antigen A contained 0.5% phenol.
** Antigen B prepared without phenol.

.43-

Discussion: The coagulative effect of concentrated dilutions
of phenol on proteins is well known, so it might be anticipated that by
the omission of phenol from the agglutination system, any precipitation
resulting from this property of phenol would be absent. An examination
of the table shows that precipitations occurred in non-phenolised antigens
to as great a degree in two lots of sera, as in phenolised antigens, and
that on the other lot the number of sera clouding in the former was almost
as great as in the latter. It does not appear therefore that a concentra-
tion of 0.5% phenol in the agglutination system exerts much of a coagula-
tive effect, or that it is because of this supposed effect that clouding
occurs. Further, since non-phenolised antigens showed no demonstrable
superiority over phenolised antigens in preventing clouding, it does not
appear desirable to dispense with the preservative effect which a 0.5%
concentration of phenol confers.

Conclusion: The results here presented do not confirm Rebrais-
ser's view that omission of phenol is an efficient means for preventing
cloudy reactions.

INVESTIGATIONS ON RELATION OF PROTECTIVE COLLOIDS, DICREASED

VISCOSITY, FAT SOLVENTS, AND INCREASED ELECTROLYTE CON-

TENT CF ANTIGEN ON CLOUDI‘IG

The further investigations projected to try to find a means of
preventing clouding were designed so as to take recognition of the fact
that a colloidal is involved in the preoipitations which cause clouding.
It is recognizedig that the presence of certain colloids in a hetero-
genous mixture will protect other colloids contained in that mixture
from.being precipitated; this led to a line of investigation wherein

colloidal substances were incorporated into standard antigen in con-

trolled quantities and these antigens were used against chicken sera

-44-

which had produced a cloudy reaction on standard antigen. Moreover, it

is recognised that alterations in viscosity of a medium, influence the

free movement of suspended particles. Thus an increase in viscosity inter;
feres with the close approximation of particles to each other which is
necessary for cohesive force to become operativelé. .Since the clumping

of bacteria during specific agglutination is dependent on the operation

of cohesive forces and since the gathering or precipitates into granules

or flocculent form in the cloudy reaction is dependent on such forces also,
it was conceived that at certain degrees of viscosity of the antigen the
tendency for the formation of macroscopic particles might be selective

so as to permit agglutination to proceed yet retarding or inhibiting the
clouding reaction. Suitable antigens were prepared to determine the effect
of increases in viscosity of standard antigen on the appearance of the
clouding reaction. Again, if, as reported by Hitchner2’(supra), the con-
stituents of chicken serum which are responsible for clouding are fatty

in nature, a conclusion which also finds support in microchemioal tests
reported earlier in this paper, it becomes conceivable that the addition
of a fat solvent to antigen might operate to hold in solution and inoper-
ative the element responsible for the appearance of the clouding reaction.
Suitable antigens were prepared using acetone as the fat solvent and these
were put to test against various sera. Finally, the influence of electro-
lyte in varying quantities in the antigen was investigated. Northrop and
DeKruile.repcrt that increases in electrolyte content of a medium beyond
certain concentrations serves to reduce cohesive forces as between sub-
stances in that medium. Investigations in this direction were undertaken
using sodium chloride as the electrolytic substance. Since the modes of
procedure differed somewhat in the several cases the same are described

separately for each series of tests.

For the sake of preliminary trials on the various substances
introduced into antigens the same chicken sera were used so that results
might be more readily interpreted on a comparative basis. These are des-
cribed at this point:

Serum 340 - A non-agglutinating serum which gave the diffuse
type of clouding on standard antigen.

Serum 344 ,- A non-agglutinating, decidedly roily serun, which
gave the flocculating type of clouding on standard
antigen. ,

Serum 298 - A clear serum which gave the granular sedimentation
similar to true agglutination on standard antigen.

Effect of Increased Colloid Content of Antigen on Qiouding

 

Normal beef serum, gelatin, and peptonet were the colloidal
substances incorporated into standard antigen in order to determine the
effect of such modification of the test fluid on clouding. The last
named, peptone, admittedly is not a true colloid. Peptone is a hetero-
genous mixture of mono-, di-, and poly-peptides, but contains also pro-
teoses and albumoses; it is not completely diffusible, thus, it is on
the border line with regard to some of its constituents between crystal-
loids and colloids. luoreover, it lended itself to making a rather repre-
sentative series of antigens containing substances all of animal origin
in which, beef serum was a practically pure true colloid with large
molecular structure, gelatin a true colloid in.which the molecules are
smaller, and peptone a border-line substance with still smaller molecular
configurations.

Bovine Serum: Normal bovine serum was introduced into three quan-

 

tities of antigen, so as to yield a 50%; 20%, and a 10% serum antigen.
The H ion concentration of these were found to be 7.6, 7.4, and 7.2 re-
spectively. Care was observed so that the resultant density in each

oase'was comparable with tube No. l McFarland nephelometer. The antigen

 

* Difco Bacto Peptone, Digestive Ferments 00., Detroit.

~46-

7|. 11"!

contained besides bodies of Salmonellagpullorum 0.5% phenol and 0.86%1NaCl.

 

Using each of these antigens, a series of multiple serum dilutions cover-
ing the range of 1-20 to l-1280 was made with each of three chicken ears.
The results as observed at the completion of 48 hours incubation at 37° C
are given here in a summarised form.

Summary of Results in Testing Concentrations of Bovine Serum on
Clouding. Serum Nos. 340, 344, and 298.

Bovine Serum .A rather uniform settling out of the antigenic suspension
was observed in all the tubes in the series. The super?
50% natant fluid had a tubidity of 0.25 on the nephelometer
scale. Upon agitation and dispersion of the precipitate
20% through the supernatant fluid, a graded turbidity was
observed decreasing in opacity fran the higher to the
10% lower serum concentrations. Controls also were settled

out, but upon agitation yielded the original turbidity
of No. l on the mcFarland nephelometer scale.

Gelatin: Gelatin was dissolved in phenolised saline, cooled, and
added to three lots of antigen so as to yield solutions containing besides
0.85% NaCl and 0.5% phenol respectively 0.5, 1.5, and 4.0% of gelatin; the
H ion concentration of these were found to be 7.4, 6.6, and 6.0 respective-
ly. The antigens were each of a density comparable to tube No. l McFarland
nephelometer. Other matters of procedure were identical with those employ-
ed on normal bovine serum containing antigens. The results secured on this

series of tests are also given in summarised form.

-47-

Summary of Results in Testing_Concentrations of Gelatin
on Clouding_

 

 

Serum 340

Gelatin 0.5% - Fine suspension and settling in l/20 and 1/40;
None thereafter.
1.5% - Heavy suspension with some settling in 1/20 to
1/80; ending at 1/160.
4.0% - Heavy suspension with some settling up to 1—640.

Serum 344

Gelatin 0.5% - Suspension with slight settling through 1/320;
none thereafter.
1.5% - Heavy suspension with settling through 1/320;
none thereafter.
4.0% - Heavy suspension with slight settling through l/leo;
none thereafter.

Serum 298
Gelatin 0.5% - Settled out and clear through 1/320; partial at
1/640; none thereafter
1.5% - Settled out and clear through 1/640; partial at
1/1280; none in control.
4.0% - Fine suspension and maximal setting through l/1280;
none in control.

Peptone: Peptone as employed in the studies here presented on
test on materials with regard to their effectiveness in inhibiting cloud-
ing was added to antigen and utilized in the test in a manner entirely
comparable to that described for gelatin. The H ion concentration of the
0.5, 1.5, and 4.0% peptone antigens were the same, namely 7.2. Summarised

results follow.

-48.-

Summary_of Results in Testing Concentrationg_of Peptone
on Clouding

 

 

Serum 344

Peptone 0.5% - Suspension and slight settling to 1/80;
none thereafter. -
1.5% - Suspension and slight settling to 1/80;
none thereafter.
4.0%.- Suspension and slight settling to 1/80;
none thereafter.

Serum 340

Peptone 0.5% - Suspension and slight settling in 1/20;
none thereafter.
1.5% - Suspension and slight settling in 1/20 and
1/40; none thereafter. '
4.0% - Suspension and slight settling in 1/20 and
1/40; none thereafter.

Serum 298

Peptone 0.5% - Settling and clearing complete through.l/320;
, partial in l/640; 1/1280 no change.

1.5% - Settling and clearing complete through 1/320;
partial in 1/640; 1/1280 no change.

4.0% - Settling and clearing complete through 1/160;
partial in 1/320; l/1280 no change.

Summary of the Effect of Colloids

 

While the number of sera utilized in this series of determina-
tions on the effect of the addition of colloidal substances of animal
origin to antigen for the purpose of preventing non-specific precipita-
tions is small, the results are clear out and indicate that with the
technique employed it is not to be anticipated that a solution of the

problem of clouding may not be found in this direction.

-49-

EFFECT OF INCREASED VISCOSITY (F ANTIGEN 0N CLWDING

In order to determine whether a greater viscosity of the anti-
genic suspension would tend to inhibit the aggregation of precipitates by
the clouding factor, antigens containing 0.5, 1.5, and 5.0% glycerine, as
well a:g0.85%'NaCl and 0.5% phenol were made. These contained sufficient

quantities of the bodies of Salmonella pullorum to yield a density corres-

 

ponding to No. l on the McFarland nephelometer scale. The H ion concentrar
tion of these antigens was found to be 7.2 as determined by the colorometric
method.

Using each of these antigens, a series of multiple serum dilu-
tions covering the range of 1-20 to 1-1280 was made with each of three
chicken sera. The results as, observed at the end of 48 hours of incuba-
tion at 37° C are given here in summarised form.

Summary of Results of Increasing the Viscosity_of the Antigenic
Suspension _y the Use of Glycerine

 

Serum 344

Glycerine 0.5% - Suspension and settling out through 1-160;
none thereafter.
1.5% - Suspension and settling out through 1-80;
none thereafter.
5.0% - Suspension and settling out through 1-40;
slight suspension 1-80, none thereafter.

Serum 340

Glycerine 0.5% - Suspension and slight settling in 1-20 and
1-40; none thereafter
1.5% - Slight suspension and sl. settling in 1-20
and 1-40; none thereafter.
5.0% - Slight suspension and s1. settling in 1-20;
trace of both in 1-40; none thereafter.

Serum 298

Glycerine 0.5% - Settling out which scales off in quantity from
1-20 to 1-640; none thereafter.

1.5%.- Settling out which scales off in quantity from
1-20 to l-640; none thereafter.

5.0%.- Settling out which scales off in quantity from
1-20 to 1-640; none thereafter.

Conclusiong: An inspection of the summarized results shows that

 

in none of the concentrations of glycerine antigens was clouding inhibited
in the range of the usual serum dilutions employed in the routine agglutin-

ation test for S._pullorum infection. A solution of the problem does not

 

seem to lie in the direction of making the antigenic suspension more viscous.

EFFECT or ADDITION cs A FAT SCIVENT, ACETONE, T0 ANTIGEN
0N CLOUDING

Preliminary tests to determine the effect of the addition of
acetone to antigen on clouding were made by setting up in.mu1tip1e dilu-
tions of 1-20 to 1-1280, sera 340, 344, and 298, (see section above for
notations on sera) using for each serum antigens containing 0.5, 1.5, and
5% acetone. The antigens in each case were made of a density to corres-
pond to McFarland nephelometer tube No. 1, and contained 0.85%.NaCl and
0.5% phenol. The H ion concentration of the antigens, as determined by
colorometric standards was 7.0. Incubation at 37° C. for 48 hours was
employed, and final readings were made at 48 hours. The results of this

series of tests may be summarised as follows:

Serum 340

Acetone 0.5% - No increase in turbidity - no floccules or sediment.
1.5% — No increase in turbidity - no floccules or sediment.
5.0% -‘No increase in turbidity - no floccules or sediment.

Serum 344

Acetone 0.5% - Floccules and sediment through to 1/160 dilutions;
' no changes in higher serum dilutions.
1.5%~- Floccules and sediment through 1/160 dilutions; no
change in higher serum dilutions.
5.0% - Floccules and sediment through to 1/80 dilution; no
change in higher serum dilutions.

Serum 298

Acetone 0.5% - Settled out and clear through 1/640 as in complete
agglutination; no change in higher serum dilution.

1.5% - Settled out and clear through 1/640 as in complete
agglutination; no change in higher serum dilution.

5.0% - Settled out and clear through 1/640 as in‘oomplete
agglutination; no change in higher serum dilution.

 

 

rilllillu‘l

.Illl...

l.ll|| i|lll\

While clouding appeared to be inhibited in the case of serum 340
and appeared only in the denser dilutions of serum 384 yet judging by the
results as secured on serum 298 it became apparent that if acetone were to
serve as a means of preventing clouding, a higher percentage of the same
probably would be required. Accordingly antigens were prepared contain-
ing up to 25% acetone, and these were used with l to 50 dilutions of four
sera selected as representative of types frequently encountered, viz:

Serum 2768 - An agglutinating serum, penny hemolysed and

showing a curdled precipitate.

Serum 2741 - A.clear agglutinating serun.

Serum 2765 - A non-agglutinating clear serum which gave the

clouding reaction on standard antigen.

Serum 2779 - A non-agglutinating, non-clouding, clear serum.

The antigens used corresponded in density, and in NaCl, and
phenol content with standard antigen. The only difference in preparation
was in the introduction of acetone in place of a commensurate volume of
water. In Table No. 19 the final or 48 hour readings after continuous
incubation at 370'was given. .

The results secured as reported in this table led to the antici-
pation that 10% acetone incorporated into standard antigen might give a
solution to the problem of inhibiting clouding. At this concentration of
acetone, agglutination though somewhat atypical because of the large size
of particles, was not inhibited, while clouding sera did not cause precipi-
tations. In order to amplify the number of cases investigated 146 non-
agglutinating sera were put to test in a 1-50 dilution in standard antigen,
and in standard antigen containing 10% acetone. After 48 hours' incuba-
tion at 37° C the readings when.tabulated showing the following comparative

results on the two antigens.
Unchanged Turbidity '

Standard antigen I Clouded 110 ' 36
10% acetone antigen II " 105 41
Reaction agreeing on both ' 98 34
No clouding on I, but clouded on II 5
No clouding on II, but clouded on I 9

i.

 

 

 

Table No. 19.

 

Effect of Concentration of Acetone upon the Clouding and

Agglutination Reactions

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

25% 207: 15% 10% fl 5% 2.5% _ .
: : 3 3 3; 3 : f : : g ;
:E : :5 : g3 : = - : : . .
{3 a: r” q: :+2§: : : . ~ - -
so. es. en. : g z : z =
.M M a :0 "4. I‘d +3 . O o .
:1: g: :3 +5: H; 3: ° : = - . ' ‘
Cloudiqg ;3 g; Clouding a3 g; Clouding =252= Clouding : . Clouding : : glouding f I
Serum ; Reaction :fifih' Reaction :gyfi: Reaction‘: :3? : Reaction - : Reaction ' :q sac: gn.T 5‘? :
Number :Susp.:Sed.gTurb.f‘ :Susp.;Sed.:Turb.:< rfiuspfzsed.:Turb.:__g38usp.:8ed.: Turb.: :Suap,:Sed.:Turb.: :susp..se " ur '° °
1
2758 + +1 - e + t1 1* e - a 1+ , - - Tr — - Tr - ' Tr
1 1
2765 + + 1 ~ + + 1+ - + . 1. _ _ _ 1 - - 1 - - l
2779 .. '1' 11- .. - Tr 1+ .. .. .. Tr _ _ _ 1 .. .r 1 .. -- 1

 

l - Precipitete of large floccules - atypical of agglutination.
2 - Sediment lacks cohesion of tvpically agglutinated particles.

V

-53-

 

Conclusions: While the preliminary trials on a small number of

 

sera indicated that acetone might be an efficient agent in preventing cloud-
ing, the expected results were not secured when a larger nmber of sera
were tented. It is therefore to be concluded that acetone in the manner

used is not effective in inhibiting the clouding reaction.

EFFECT CF VARIOUS CONCENTRATICNS CF THE ELECTROLYTE, Na01, 0H CLGJDING

Data were secured by means of preliminary tests with antigens
containing 10.0, 5.0, 2.5, 1.25, and 0.625% NaCl (Baker, c.p.) on .1: cloud-
ing non-agglutinating sera, five agglutinating, non-clouding sera, and one
non-agglutinating, non-clouding serum. These antigens had a density cor-
responding tc tube No. l of the tharland nepheluneter scale and contained
besides the respective percentages of sodium chloride, 0.5% phenol; their
H ion concentrations were determined to be 7.0 by the coloranetric method.
The serm dilution in each case was 1-50. Final readings after 48 hours'
incubation at 37° C are presented in Table No. 20.

Fran analysis of the data thus secured 1.25% to 5.0% NaCl antigens
appear to be effective in preventing non-specific precipitations; in the
10.0% and 0.625% NaCl antigens clouding appeared. None of the concentra-
tions of NaCl employed inhibited agglutination.

In order to secure further data on the effectiveness of a 5%
Na01 content of antigen in inhibiting clouding, tests were run on 146
sera, simultaneously using the same sera on standard antigen. Both quan-
titiee of antigen contained 8. pullcrum in sufficient quantities to yield
a density as of Tube No. 1 McFarland nephelcmeter and also 0.5% phenol.
The serum dilutions employed in each case were 1 to 50; incubation was at
37° 0 for 48 hours. The final or 43 hour readings are presented in the

following summarised form:

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Turbidity

Clouded Unchanged % Clouded
Standard antigen I no so ' 75.3 -
5% 1.01 antigen II 2 144 1.3 -
Reaction agreeing on both 2 36 - - -
Clouded on I, not on II -- -- - - - 108
CloudedcnII,notcnI -- .. -.... 0

Discussion: The results secured indicate that NaCl in the con-

 

centration of 5% in standard antigen has a marked inhibitory effect on the
appearance of clouding. It should be noted that in the preliminary tests
given earlier in this section 2.5 and 1.25% 119.01 gave equally good re-
cults in the inhibition of clouding. It i. to be expected that 5.0% llaCl
may not be the optimum concentration of the salt in antigen to produce
the desired effect but that the optimum lies sanewhere between 10.0 and
1.26%

The results secm'ed are sufficiently conclusive to justify the
assertion that the suppression of clouding in a high percentage of sera
may be effected by making up the antigen for the agglutination test to

contain 5.0% NaCl.

 

Comparison of the Efficienm of NaCl AgtTigen andlag
Antigens inthe hibiticn of C oudlnjg

 

 

Mathews (supra) reports that an antigen made of E. gallinarum
in 0.5% phenolised, normal saline to a density corresponding to tube
lo. 3 of the McFarland nephelcmeter scale, and to which is added 2 mils
of a 2% sodium hydroxide solution is efficient in inhibiting clouding.

Stafseth‘ gives confirmation to the effectiveness of this agent.

 

e Personal ccununication.

_RA._

In order to learn whether sodium hydroxide would be effective
in an antigen of the density used throughout the course of this study,
namely, one corresponding in density to tube No. 1 of the McFarland scale,
a series of duplicate tests were run simultaneously on standard antigen
for a control series; on 5% NaCl antigen; on ma: antigen of a density of
lo. 1; and on NaCB antigen of a density of No. 3 on the McFarland scale.
The H ion concentrations of these antigens were respectively 7.2, 7.0, and
7.2 for both of the Nam antigens, as determined by the coloranetric
standards, when put on test. The incubation was at 37° 0 for 48 hours.
The results on the 65 clear, non-agglutinating sera follow.

Thirty-one (31) sera produced clouding in the standard antigen.
Ten (10) sera produced clouding in the Real antigen of density No. 1; two
of these sera had not produced clouding on the standard antigen; the type
of clouding noted in all ten cases was that of a flocculent sedimentation
with the supernatant liquid water clear. One sense out of the 65 clouded
the lam antigen of density No. 3, and the type of clouding in this case
was a flocculent sedimentation, with the supernatant liquid water clear.
In the 5% Heel antigen no clouding occurred except that the same serum
which produced a flocculent sedimentation in the New antigen cf density
lo. 3, produced a slight diffuse clouding, which gave a reading of No. 2
on the McFarland scale.

Discussion: Mathews reports that the use of sodium hydroxide

 

is attended with sue risk of disturbing the antigenic suspension of _E_._
ellinarus by causing a loss of sensivity, and recasmsnds its use only when
the condition of the sera shows that clouding may be expected. It is con-
ceivable that when a lesser nunber of bacterial bodies are present as is
the case in an antigen of No. 1 density, the clouding factor may operate

because of the absence of sufficient nunbers of those bodies even in the

presence of equal concentrations of Nam. This deduction probably is
valid in the light of the conclusions drawn from the work on the source
of the clouding factor presented in this paper, and seems to explain
the failure of the antigen of §34pullorum of density No. l with Haflfi
added, as compared with the other NaOH antigen, where the results were
more favorable.

For it was clearly shown in that section of this paper that,

a dense suspension of antigen in itself tends to inhibit clouding, and
that clouding interference in antigens of a density of either tubes

three or five of the McFarland nephelcmeter is markedly suppressed. that
the addition of Nam to antigen as recmended by Mathews was not without
inhibitory effect upon the cloudy reaction is supported by the observa-
tion here made that on the sixty-five sera.examined thirty-one gave this
reaction on standard antigen while only,ten gave it on standard antigen,
each 100 o.o. of which contained 2 mils of 2% Nam. Part of the inhibitory
effect exerted by antigens prepared as recommended by this investigator
must, however, be ascribed to the increased numbers of bacteria contain-
ed in such antigen. This is supported further in this'work'by the fact
that only one serum in sixtybfive gave the cloudy reaction on a density
lo. 3 EcFarland nephelueter antigen, while ten produced it on a density
No. l, the content of the antigen in Nam, phenol, and NaCl being the
same in both cases.

It would have been interesting indeed had opportunity been
offered to necropsy and culture the ten birds giving the cloudy reac-
tion on the density No. l Nada antigen and the bird giving that reac-
tion on a density No. 3, NaOH antigen, the reactions in all these cases
resembling a positive specific reaction so that a differentiation could

not be made. For none of these birds gave a positive specific reaction

on standard antigen, nor on density No. l, 5% NaCl antigen. The relative
diagnostic accuracy of these various antigens is a very pertinent ques-
tion but must for the present go unanswered.

gqnclusions: As between the antigens under test, the 5% NaCl

 

antigen showed a superior efficiency over the NaCfl antigens in inhibit-
ing clouding.

‘ The qualities of an antigen such as suggested by Hathews in
inhibiting the cloudy reaction must be ascribed in part to its New con-
tent, and in part to its greater density; its diagnostic accuracy however

may be questioned .

A Note on the Plate Test Methgd and Cloudigg

 

1
Following the appearance of a paper by Huddleson—g on the plate

test method for testing sera of cattle for the presence of Bacterium abor-

 

t_u_s_ agglutinins, Runnels, Coon, Farley, and Thorp-l-a reported an adapta-
tion of that method to testing for S. pullorum infection. Using the methods
described by these writers the plate test method was put to trial on a num-
ber of clouding and agglutinating sera. It was found that by the plate
test method clouding react ions appeared as rapidly as agglutinating reac-
tions and that it was difficult to distinguish between the two. This is

in agreement with what one would expect judging by the results given in
Table No. 20. Where 10% NaCl antigen was used on clouding and agglutinat-
ing sera, floccules of precipitate appeared here in both types of sera,
the only difference being in the size of particles and possibly in abund-
ance. In the plate test method a 12% solution of NaCl in an antigen of

a density 50 times as great as 0.75 to 1.0 on the McFarland nephelaneter

scale is used. In this type' of antigen, the plate method yielded preci-

-59-

pitates which were slightly more abundant and in somewhat larger parti-
cles on agglutinating sera than on clouding sera. Yet the differences

‘were not sufficiently marked to permit a reliable differential reading.

SECTION V
BACTERIOLOGIC AND PATHOLOGIC FINDINGS ON BIRDS GIVING CLOUDY OR
POSITIVE REACTIONS TO THE AGGLUTINATION TEST FOR
SALMONELLA PULLORUM INFECTION

In this section there is presented a report on a group of birds
selected from three farm flocks which reacted positively or clouded on the
routine agglutination test. It was deemed desirable to obtain a check
on the accuracy of the reading of the test, on the differential diagnosis
of the reactions called ”positive" or "cloudy”, and on the efficiency of
the antigen employed by'making appropriate bacteriological tests and ob-
serving pathologic conditions at necropsy.

Protocols and original photographs accompany the individual
case records, of representative types of lesions encountered. Photographs
were made only of those cases which presented a pathologic picture differ-

ing from those previously encountered during the course of the work.

Methods Used in Culturing the Ovaries and other Organs

 

The birds were killed by bleeding from within the throat, using
the commercial sticking method for a dry pluck, and blood samples were
taken to discover the titre of the serum at necropsy. The feathers were
removed and with the bird laid on its back, the viscera were exposed by
‘making an incision through the abdominal wall in the pelvic region and
cutting through the entire body wall on both sides of the abdomen and
thorax following the Junctions of the vertebral and eternal portions of
the ribs to the coro-co-scapulo-clavicular articulation by means of
sterile shears. This permitted breaking back.the entire floor of the
abdomen and thorax. The viscera were then examined for gross patholog-

ical lesions.

The entrails were drawn aside to expose the ovarian mass. Cul-
tures of suspicious looking ova were made either by scaring the surface
of the ovum, and then puncturing it so that the inoculum might be secur-
ed, or by tearing out the selected ovum with sterile forceps, and crushp
ing it with them after transfer to the agar slant. Inoculations were made
onto several standard agar slants and into standard broth, which medias
were of pH of 7.0. The incubation period was at 37° 0 for free 12 to 36
hours, depending upon the luxuriousness of the growth.

After having made the inoculations, detailed notes were taken
of the conditions present in the cadaver. Cultures of other organs and
of the oviduct were taken by the first method. Where any of the tissues
had been exposed to air contamination, they were scared before securing
the inoculum. Where procedures differed frm these, a notation to that

effect will be found in the case reports.

Bacteriological Tgchniqm
When more than one type of growth resulted on. the initial trans-
fer, plating out by the agar tube dilution method was used to secure pure
cultures. In a number of cases this was not necessary, as well isolated
colonies were secured on the original transfer, which by subsequent tests

were found to be S. pullorum.

 

The pure cultures secured were subjected to a study of their
morphology, reaction to Gram stain, agglutinability by means of 8. ml-
lorum anti-serum, and their fermentation reactions on various carbohyd-

rate media. Identification of the strains as S. pullorum was considered

 

establist when examination showed them to be Gram-negative, non-spore-
forming, non-motile, singly occurring rods, in size less than 1 micron

in width and ranging free: 1 to 2 microns in length, which were agglutin- .

ated by S. pullorum anti—serum, and which fermented Dextrose, but not Dul-

 

citol, Lactose, Maltese, and Sucrose.

The carbohydrate media were prepared as follows. Sterilised
broth containing 0.3% Armour'e Beef r3xtract, 1% Difco Peptone, and 0.5%
Sodium Chloride (Baker, c.p.) was inoculated with a 24 hour broth culture
of Escherichia communigg_and incubated for 24 hours. It was then boiled
and filtered through filter paper. Difco Bacto-Agar to yield a concentra-
tion of 0.5%‘was then added, and the pH reaction adjusted to 7.0. One per
cent of Andrade's indicator was added prior to tubing and sterilizing the
agar. To the resudtant semi-solid agar medium, while cooling after steril-
isation, quantities of 20% aqueous solutions of the carbohydrates, which
had been sterilized by filtration through.Berkefeld filters, were added to
yield a concentration of 0.5%»in the final medium. Carbohydrate semi-solid
agar slants were thus prepared containing Arabinose, Dextrose, Galactosc,
Levulose, Hannitol, Mannose, Rhamnose, Xyloee, Dulcitol, Lactose, Kaltose,
and Sucrose (Pfanstiehl). They were incubated for 24 hours at 370 C to
test their sterility, and then sample tubes were checked for characteristic
fermentation by standard cultures.

Readings of the reactions were made at the end of 24 and 48 hours,
and the results secured on the various strains isolated are presented in
Table No. 21.

Discussion: It may be noted that May and Goodner-g-g- in a study

 

of several groups of strains state that S. pullorum ferments Xyloee media.
None of the strains isolated in‘the course of this study fermented this
sugar. The explanation of the discrepancy may lie in the fact that all

of our strains had been grown in laboratory media for a considerable period
of time, whereas these strains had been recently isolated. It is recognis-
ed that organisms do very in their biochemical reactions, and that they

make adaptations under artificial conditions of environment.

Table No. 21

 

Fermentation of Various Carbohydratest by Strains of S. pullorum
Isolated During the Course of this Study.

enemasmm

access:

accesses.

eccasboq

enovocdsm

enoavkon

ecosapsnd

 

Cultures
from
Case

Hum

.. . .. .._

new
«04

use
40¢

use

wed

ecu

«o4

2.;

sec

and

3;

new

«ed
:a

new

«04

 

 

O. O. .0 .0 I. O. O. O. O. .O O. C. O. O. O.

A.AA.AA.-—AAAAA

O. O. .0 I. C. O. C. O. O. O.

.G-GG-.G-G-G-G
aaaanaaa.aaaana

.0 e. e. O. .0 00 I. e. e. e. 00 O. O. .0 O. O.

.G--.-GGGG..

i

.A.AA..AAAA.AA.

O. O. O. O. O. O.

.0..-

naoaea..0-

.AAAA-AAAAAAAAA

ee e e 00 e es 0. e. e. e. 00 ee 0. e.

«.m.wnunm ununu.ununu .nununu

AAAAAAAAAAAAAAA

00 ee ee 00 ee ee 0. es 00 ee 0. es s. 00 ee ee

GG.GGGGG.GGGGGG
AAAAAAAAAAAAAAA

00 ee 0. 00 O. 00 e. es 00 ee 0. so e0 00 e. s.

...G.G...GG.GG.
.A ..A.A.A.A.A . ._A.A.A.ATA_A

so so so so so so as so so es so es s. 0. es es

12345678901‘789
111111

0. O. .0 O. O. .0 O. O. O. O. O. O. O. 0. O. O.

 

included in this tdble, as none of these were fermented

s xylose, Dulcitol, Lactose, Maltese, and Sucrose are not
by these strains at the time of their isolation.

 

“I‘ll" all. 7: Mail

IL

.4 a...

 

The other carbohydrates were fermented with varying degrees of
regularity, so that no general statemnt can be made respecting any of
the strains, save that all fermented Dextrose and Galactose, and did not
ferment Xylose, Dulcitol, Maltese, Lactose, and Sucrose. The other carbo-
hydrates used placed in decreasing order of fermentability by these strains
were Mannose, Levulose, Arabinose, .Rhamnoee, and lannitol.

Of the 15 cases which were diagnosed as positive on the routine
test, two had agglutinated the 1-50 dilutioncaapletely only after 72
hours of incubation. At necropsy the organism was recovered from these
cases (8 and 9), which would tend to show that any routine test which
is based on incubation at room temperature overnight as is the practice
at some stationeg'l, or on a 24 hour reading after incubation at 37° 03'
where agglutination in a l-5O dilution is used as the diagnostic titre
will fail to condemn all the birds tested which actually harbor the infec-

tions

Salmonella pullorum was not recovered from any of the four cases

 

which gave cloudy reactions, where it was thought that clouding or non-
specific precipitation might have mashed a true reaction and thus failed
to condemn the bird.

A few miscellaneous organisms were recovered fras ease of the

cases, and are mentioned under the respective case reports.

Conclusions: Salmonella pullorum was not isolated from the

 

birds studied which gave cloudy reactions. This organism was recovered
in every instance, from necropsies" on birds diagnosed as positive on the
ugglutination test.

Incubation at 37° for 24 hours, or incubation at roan temper-
ature overnight would fail to reveal all birds harboring the infection

‘hen a l to 50 titre is used as for diagnosis of infection.

-55-

There is a variability in the fermentation reactions of freshly
isolated strains on carbohydrate media, except that Dextrose is fermented

regularly and that Dulcitol, Lactose, maltose, and Sucrose are not fermented.

CASE NO. 1
Bistosy:

White Leghorn hen frcfi Flock No. 1. Serum agglutinated standard
antigen on routine test 4 1- and l e in the 1-50 and l-lOO dilutions respec-
tively at 48 hours. Killed 1 week after test.

Examination shows the bird to be heavily fleshed. The legs, beak,
and vent are yellow and bird is apparently not in lay.

Necropgy{Findingg:

There is a large amount of fat about the viscera. The liver is
congested and friable. The ovarian mass is much reduced in size. There
are several pedunculate ova of irregular shape which are grayish to bluish
green in color and whose contents are caseous. There is a small cyst ad-
jacent to the ovary measuring 4 m.m. in diameter. The cystic fluid is
clear.

Serum TitAr‘g:

 

4 c- in the l-4O and 2c in the 1-80 dilutions at 48 hours.
Bacteriological Finding;

Salmonella pullorum was isolated from the necrotic ova. This

 

strain was agglutinated by positive sera 4 e in the 1-160 dilution. Acid
and gas reproduced on Dextrose; acid only on Arabinose, Galaotose, Mannose,
and Rhamnose; neither acid nor gas on Levulose, hiannitol, Xylose, Dulcitol,

Lactose, Maltese, and Sucrose.

.1...1I.E.!..;IL.. I. I, one
anti 7 f...
._

Ia

 

Bacteriological Findingg:

Stgphylococcus albus was isolated from the albumin secreting

 

portion of the oviduct.

CASE NO. 2
Hilton:

White Leghorn hen from Flock No. 1. Serum agglutinated standard
antigen in routine test 4+ in both the 1-50 and l-lOO dilutions at 24
hours. Killed 2 weeks after test.

Examination shows bird to be well fleshed and apparently not in
lay.

Necropsy;Findingg:

There is considerable visceral fat. Liver and heart appear nor-
mal. There is a degenerated ovum, yolk ooncretion, 4 m.m. in diameter ad-
herent to the mesentery. Another degenerated ovum 5 by 10 m.m. shaped
like a peer, is embedded in an indurated fold of the mesentery; it is
mottled yellow and brown in color. The ovarian mass is comparatively
small; the largest ovules are 3 to 4 m.m. in diameter and are of a pale
yellow to bleached color.

Serum Titre:
3 e in the l-40 and 2 c in the l-80 dilution at 48 hours.

Bacteriological Findings:

 

Salmonella pullorum was isolated from the yolk concretions.
This strain was agglutinated by positive sera 4c in the 1-160 dilution.
Acid and gas are produced on Dextrose, Galactose, Levuloee, hannitol, and
Hannose; neither acid nor gas are produced on Arabinose, Xylose, Dulcitol,
Lactose, Maltese, and Sucrose.

Staphylococcus albus was isolated from the oviduct.

Cultures of crushed immature ova remained sterile.

I 67-

Ilvll. «I . v I.
lall-sli|. ... .alftl} .. fdu

sac... _ .- -

 

CASE NO. 3
Histog:

White Leghorn hen from Flock No. 1. Serum agglutinated standard
antigen in routine test 4.- in both the 1-50 and 1-100 dilutions in 24
hours. Trap nest record shows bird to be a high producer and in lay at
time of test. Killed 2 weeks after test.

Examination shows bird in early moult. Has pale legs, pale beak,
and pale vent. Comb is pale to powdery.

Necropsy‘Findings:

General: Diffuse peritonitis is present, and a cloudy'brownish
fluid fills the abdominal and pelvic cavities. vessels of the mesentery
and peritoneum are injected. The mesentery is marked by nodules which have
the glistening appearance of fat throughout its extent. These nodules
present a beaded appearance and are from 6 - lO m.m. in diameter. (See
Plate II). Diagnosed as a case of lympho-sarccmatosis (Kitt).

Ovarian Mass: Four ova which are angularly ovoid, are necrotic;

 

their enveloping capsules are shrunken and roughened; in color they are a
mottled greenish-yellow to brown. They range in size from 1.8 c.m. by

l c.m. to 4 c.m. by 3 c.m. There are numerous ovules ranging from pin head
size to 4 m.m. in diameter of a pale yellow to bleached color. Remainder of
ovarian mass presents no striking features (See Plate I).

Oviduct: There is an area of cirrhosis on the oviduct, having a
depth of l c.m. and extending 3 c.m. anteriorly from the shell gland. An
indurated mass 2.5 c.m. by 2.0 c.m. by 1.8 c.m. lies adjacent to the evi-
duct and attached to it by band of hardened mesentery. On section, the
cortex is of a dense fibrotic character, with an amorphous center.

Serum Titre:

4 e at 1-160, 2 . at 1-320 at 43 hours.

-68-

IqliIIIIEI a‘fldr .. e ..

Jump”. .. _-

 

 

 

 

 

 

Case No. 3. Showing necrotic ova situated on‘both
sides of the ovarian mass. The mesentery showing
lymphosarccmatosis has been folded back. S._pullorum

was recovered (See text). (Degree of enlargement
x).

 

 

 

 

 

PLATE II

Case No. 3. The intestine with attached mesentery has
been removed to show the beaded masses distributed through-
out the extent of the mesentery. Condition diagnosed as
lymphoearccmatosis (Kitt). (Degree of enlargement %-x).

-70-

Bacteriological Findings:

Escherichia coli was isolated from the peritoneal fluid.

 

Salmonella pullorum.was isolated from the necrotic ova. This

 

strain was agglutinated by positive sera 4 e in 1-160 and 2 e in 1-320
dilutions. Acid and gas are produced in Galactose; acid only in Dextrose,
Arabinose, Hannose, and Rhamnose; neither acid nor gas in Levuloee,

Hannitcl,-Xylose, Dulcitol, Lactose, Maltese, and Sucrose.

CASE ho. 4
History:

White Leghorn hen from.Flock No. 1. Serum agglutinated standard
antigen on routine test 4.e in both the 1-50 and l-lOO dilutions at 12
hours. Killed 2 weeks after test.

Examination shows bird to be in good flesh.
gecrgps11Pindigge:

General: The oviduct is misplaced resting on the floor of the
abdominal wall. The right oviduct persists and ends in a blind sack which.
contains a loosely attached lump of irregular shape and fluid (Plate III).
There is.a pathologic enlargement near the cloaca of irregular outline
and rounded surface measuring 4.5 by 2.5 by 2 c.m. (Sarcma). It 1.
hemorrhagic and has several loosely attached gray to blackish masses on
its surface which have the appearance of disintegrated ova. The mesentery
is injected.

Ovarian Mass: At the lower border of the ovary are two large
pedunculate ova measuring 3.5'by 3 by'z c.m. which because of the constric-
tion of the enveloping capsules present a lobulated appearante (See Plate
IV). They are injected and are of a dirty greenish yellow color. In one

of these the capsule is shrunken and roughened (Coagulation necrosis).

 

 

 

PLATE III

Case No. 4. Shows double oviduct; the right ends in a
blind sack which contains a neoplasm and fluid from.which
S. pullorum was isolated. (Degree of enlargement 2/3 x).

 

-72-

 

 

 

PLATE 1V

Case No. 4. Shows the large necrotic and lobulated ova,
with shrunken and roughened enveloping capsules, as well
as the pedunculated type with caseation necrosis. S.pul-
lorum was isolated. (Degree of enlargement l x).

-73-

One ovum 2.2 c.m. in diameter of a dark orange color which is petechiated
and injected lies at the upper border. There are ten pedunculated ova,
angularly spherical to irregularly ovoid in shape, ranging in size from
8 m.m. to 1.7 c.m., all of which are a mottled green and yellow color and
hate caseous contents. Many are petechiated and injected. There are
several areas of necrosis among the immature ovules. The dorsal ovarian
ligament is indurated.

Serum Titre:

 

4 e in the 1-640 dilution at 48 hours.
Bacteriological Finding :

Salmonella pullorum was isolated frees the necrotic ova. This

 

strain was agglutinated by positive serum to 4 e in the l-640 dilution.
Acid and gas are produced in Arabinose, Dextrose, Galactose, and Mannose;
acid only in Levuloee, Mannitol, and Rhamnose; neither acid nor gas in

Xylose, Dulcitol, Lactose, Maltese, and Sucrose.

CASE NO. 5

Histogz:

White Leghorn hen frcn Flock No. 1. Serum agglutinated standard
antigen on routine test 4 o- in both the 1-60 and l-lOO dilutions at 12
hours. Killed two weeks after test.

Examination shows bird in good flesh and apparently not in lay
judging by the yellowness of the shanks, beak, and vent.
He or opsLF ind in $3.:

Organs other than the ovary appear to be normal. The ovarian
mass measures 6 by 4 c.m. in situ. At the lower border is a pedunculated
ovum with an injected leathery capsule of a mottled dark gray and greenish

color, measuring 2.3 by 1.5 c.m. by 9 m.m. Adjoining it is another of

-74-

.lnll.‘.._.e ... ...4||.'.. numwg

a]... v... a
-

L

similar appearance but smaller. They contain an amber colored fluid
with green and yellow caseous lumps (caseation necrosis). There are
two ova which because of the constriction of the capsules present a
lobulated appearance, as though several adjacent ova had becane con-
fluent. These are of a golden yellow color and measure 1.5 by 1.5 by 1
c.m. 0n the right border is a pkedunculate ovum, measuring 1.2 c.m. by
8 by 8 m.m. of a jet black color W a dark fluid and lumps (ceagu-
1ation necrosis). There are ntmxerous ova from microscopic to pea size of
a pale straw to bleached color. Iany are injected. Plate V.
Serum Titre:

4 e in the 1-320 dilution at 48 hours.
Bacteriological Findirgs:

Salmonella pullorum was isolated frm the necrotic ova. This

 

strain is agglutinated by positive sera 4 1- in the 1-640 dilution. Acid
and gas are produced in Dextrose, Galactoee, and Hannose; acid only in
Arabinose, Levuloee, and Mannitol; neither acid nor gas in Rhamnoee,

Xylose, Dulcitol, Lactose, Maltese, and Sucrose.

' CASE NO. 6
History:

White Leghorn hen from Flock No. 1. Serum agglutinated standard
antigen on routine test 4 a in both the 1-50 and 1-100 dilutions at 12
hours. Killed 3 weelos after test.

Examination shows bird to be heavily fleshed. The shanks, beak,
and vent are yellow and the bird is apparently not in lay.
Necrcmsyj‘indiggg:

There is much fat about the visara. The liver is congested, en-

larged, and friable. (he degenerated ovum has a fibriucus attachment to

 

 

_‘.__w

 

 

 

 

Case No. 5. Shows the type of infection where
because of constriction of the capsules, it appears
as though several ova'had become confluent. Large
ovum on right is of a jet black color and firm in
consistency (coagulation necrosis). 8. pullorum
was isolated from the necrotic ova. (Digree of
enlargement 1 x).

 

the mesentery. There are four pedunculated ova of a mottled dark grayish
green color and of hard consistency measuring 1.5 by 1 c.m. by 8 m.m. Their
contents are caseous. There are several smaller ova which are also peduncu-
lated, and of the same color, that show caseation necrosis. Remainder of
ovarian mass consists of immature ovules of a pale straw to bleached color.

Senna Titre:

 

4 e in the 1-320 and 2 1- in the 1-640 dilutions at 48 hours.

Bacter ieleiio a1 F ind inL:

 

Salmonella pullorum was isolated fran the necrotic ova. This
strain is agglutinated by positive sera 4 e in the 1-320 dilution. Acid
and gas are produced in Arabinose and Dextrose; acid only in Galactose,
Mannose, and Rhamnose; neither acid nor gas in Levuloee, Mannitol, Xylose,

Dulcitol, Lactose, Maltese, and Sucrose.

CASE NO. 7

History:

White Leghorn from Flock No. 1. Serum agglutinated standard
antigen on routine test 41- and 31- in the 1-50 and 1-100 dilutions at 48
hours. Killed 3 weeks after test.

Examination shows bird to be well fleshed and apparently not in
lay.
lecropeIFindinL:

The ovarian mass is much reduced in size. The six largest ova

MIL

measure 6 by 4 by 3 m.m. Of these, We are pedunculated and show in-
jectien and petechiation and are of .a dirty yellow color. There are
several smaller ova 4 by 3 - 2 m.m. which have a confluent appearance

and are of a dirty yellow color and are caseous. Other organs show no

alterations frm the normal.

-77-

Serum.Titre:
Se in 1-160 and l e in the 1-320 dilutions at 48 hours.
Bacteriolggical Findingg:

Salmonellapullorum was isolated from the necrotic ova. This

 

strain is agglutinated 49 in the 1-160 dilutions at 48 hours. Acid and
gas are produced in Dextrose, Galaotose, and Levulose; acid only in
Arabinose, kannose, and Rhamnose; neither acid nor gas in Mannitol, xylose,

Dulcitol, Lactose, Maltese, and Sucrose.

CASE NO. 8
Histogy:

‘White Leghorn hen frcmfiFlock No. 2. Serum agglutinated standard
antigen in routine test 49 in 1-50, and 1+ in 1-100 dilutions at 72 hours.
Record shows that bird has been noted to lay bloody eggs frequently. Kill-
ed 6 weeks after test.

Examination shows bird to be in lay. Legs, beak, and vent are
of a pale yellow color.

NecropsyiFindings:

General: Bird is in good flesh, but not fat. No variations from

the normal are noted except in the ovarian.mase.

Ovarian.Mass: Two ova, which are spherical and of a golden yellow

 

color and whose diameters are 2.4 c.m. and 3 c.m. have capsules which are
injected and pstechiated. One ovum which is angularly avoid and whose
dimensions are 2 c.m. by 2 c.m. is constricted by the stigmen, and is of

a dirty gray color (See Plate VI). Situated immediately above the largest
ovum, and to the right of the median line is a pedunculated ovum 6 mam.
by 4 m.m. by 3 m.m. of an almost jet black color, the content of which is

caseous. ”Within the ovarian mass are several ova of the size of a scotch

-78..

 

 

 

 

 

 

 

PLATE VI

Case No. 8. Presents no marked lesions.“ maturing ova
show petechiation and injection. S. pullorum was re-
covered from the ovum showing the broad stigmen (upper
left). Agglutination was complete in 1-50 only after
72 hrs. incubation. (Degree of enlargement 2/3 x).

 

-79-

pea which are pedunculated and of a mottled gray to green color. Remainder
of ovarian.mass presents no pathologic features.
Serum Titre: I
49 at 1-80 and 2 a at 1-160 at 72 hours.
Bacteriological Findingg:

Salmonella pullorum was isolated from the fluid in the largest
constricted ovum and from the caseous matter contained in the blackish
ovum. This strain was agglutinated by positive sera 4+ and 2+ in the 1-160
and 1-320 dilutions respectively. Acid and gas are produced in Dextrose,
Galaotose, Levuloee, Mannitcl, and Mannose; neither acid nor gas is pro-
duced in Arabinose, Rhamnose, Xylose, Dulcitol, Lactose, Maltese, and

Sucrose.

CASE NO. 9
Histogz:

White Leghorn hen from Flock No. 2. Serum agglutinated standard
antigen in routine test 4+ and 2+ in the 1-50 and 1-100 dilutions respective-
ly'at 72 hours. Killed 7 weeks after test.

Examination shows bird to be in good flesh. The legs, beak, and
‘Went are yellow and the bird is apparently not in lay.

Iflzcropsy'Findings:

General: Bird is in good flesh, and there is considerable fat
about the mesentery. Organs other than the ovary show no striking features.
Trhere is a dilation of the rectum just anterior to the cloaca and extending
forward for 4 c.m. The maximum diameter of the dilation is 2.5 c.m. A
thin disk of a mixed crystalline and amorphous deposit which lies within

‘the cloaca is the probable cause of the dilation.

-80..

.— .. w *A—H‘

 

 

 

 

 

9 10 ll
PLATE VII

Ovarian masses from Case Nos. 9, 10, and 11, show
various grades of lesions from which 8. pullorum
was isolated. (Degree of enlargement 1 x7:

 

l A -8 2-

the cloaca, and the dilatation extends anterierly for 7.5 c.m. Examina-
tion shows a mixed crystalline and amorphous deposition of urates at the
sphincter ani as the probable cause of the dilatation.

Ovarian Mass: The ovary is much reduced in size, measuring 3.5

 

c.m. by 2 c.m. by 6 m.m. in situ. At the posterior border of the ovary,
are four pedunculate, degenerated ova of a coal black color. They are
roughly cubical in form, and measure 4 m.m., 5 m.m., 6 m.m., and 7 m.m.
respectively. They are very hard, and when incised present a dry caseous
appearance. Attached to the anterior border of the ovary by slender
pedicels 1.5 c.m. and 1 c.m. in length are two ova whose enveloping cap-
sules are leathery. They are gray in color, and contain a jelly-like
mass (See Fig. 2, Plate VII). There are numerous smaller ova ranging
fran pin head to small pea size, some of which are of a mottled green to
brown color.
l§erum Titie:

4e in 1-40 and 2+ in 1-80 at 48 hours.

Bacter iological F ind ings:

 

Salmonella pullorum was isolated from the necrotic ova. This

 

strain was agglutinated by positive sera 4a in 1-320 and la in 1-640 dilu-
tions. Acid and gas are produced in Arabinose, Dextrose, Galaotese, Levu-
lose, Lannitol, and Mannose; neither acid nor gas is produced in Rhamnese,

Xyloee, Dulcitol, Lactose, Maltese, and Sucrose.

CASE NO. 11
Histfl:
White Leghorn hen fran Flock No. 1. Serum agglutinated standard

antigen on routine test 40- in both the 1-50 and 1-100 dilutions at 24

hours. Killed 4 weeks after test.

 

'.

Examination shows the bird to be in good flesh and not in lay
judging by external appearances.
Necr_opsy Findi_ng_s_:

General: Bird is well fleshed but not fat. Organs other than.the
ovary show no striking features.

Ovarian Mass: This ovary shows extensive alterations due to

 

Sglmonella pullorum infection (See Fig. 3, Plate VII). Two pendulus ova
of irregular outline at the lower border of the ovarian mass are under-
going liquefaction necrosis. They are of a.mottled gray and greenish-
yellow coloration, and yield to pressure. They measure 2 c.m. by 1.5 c.m.
by 1 c.m. There are several ova irregularly ovoid in outline, 7 mwm.
to 1 c.m. in diameter which have a leathery capsule, are hard under pres-
sure, and show caseation necrosis. One degenerated ovum 2 c.m. by 1.2
c.mt by 5 m.m. is attached to the ovarian mass by a pedicel 5 c.m. in
length, and is embedded in a fold of the mesentery. It is surrounded
by a dense fibrous capsule, and its contents are caseous. One ovum with
a diameter of 9 m.m. and of a light golden yellow color is the only appar-
ently normally developing ovum in the ovarian mass.
Serum Titre:

4+ in 1-320 and 2+ in 1-640 at 48 hours.
Bacteriological Findings:

Salmonellggpullorggfiwas isolated from several of the necrotic

 

ova. This strain was agglutinated by positive sera 4+ in 1-640 and 2+
in 1-1280 dilutions. Acid and gas‘are produced in Arabinose, Dextrose,
Galaotose, Levuloee, and'Mannitol; acid only in Mannose and Rhamnose;

neither acid nor gas in Iylcse.Du1citcl, Lactose, Maltese, and Sucrose.

 

 

CASE NO. 12
History:

White Legnorn hen fran Flock No. 1. Serum clouded on standard
antigen in routine test. Killed 6 weeks after test.
Necropsy Findingg:

General: Bird is well fleshed, and there is considerable fatty
tissue about the viscera. Organs show no macroscoPic evidences of disease.
There is a bulbous enlargement of the rectum anterior to the clooca. This
dilalation measures 3.8 c.m. in diameter and is 5 c.m. in length.

Ovarian Mass: The gross dimensions of the ovary are 4.8 c.m.
by 3.6 c.m. There are two maturing ova having diameters of 1.7 c.m. and
1.2 c.m. which are of a golden yellow color. There is one pedunculated
ovum which is irregularly spherical in 'outline, of a gray-to yellowish
color, and l c.m. in diameter. The stigmen is well marked and the capsule
is corrugated, shows petecbise and is injected. Adjoining it are several
smaller ova ranging from pin head to small pea size which are hemorrhagic
and mottled gray and yellow in color. Remainder of ovarian mass present
no striking features.

Serum Titrg:

Serum at necropsy failed to cloud or agglutinate in standard

antigen.
Bacteriological Finding:
Staphylococcus aureus was isolated free: the ovum showing petechi-

ation and injection.

CASE NO. 13

Histog:
White Leghorn hen from Flock No. 1. Serum clouded on standard

antigen in routine test. Killed 6 weeks after test.

-35-

Examination shows bird to be well fleshed, and apparently not i
in lay judging from the yellowness of the shanks, beak, and vent.
NecrgpsLF ind Egg: .

The gross dimensions of the ovarian mass in situ are 4 c.m. by
4 c.mm There are 36 ova ranging in size 3 m.m. to 6 m.m. in diameter
which are light yellow, pale straw and bleached in color. A small mass
of 6 adjoining ovules situated near the ventro-posterior border of the
ovary are hemorrhagic. In size these are 1 to 3 m.m. in diameter. Re-
mainder of the ovarian mass presents no notable features; there are
numerous ovules ranging from pin head size to 3 m.m. in diameter which
are of a pale straw to bleached color.

Serum Titre:

 

Serum at necropsy failed to cloud or agglutinate standard

Bacteriological Findiggg:

Cultures from the hemorrhagic ovules remained sterile.

CASE NO. 14
Histogz:
White Leghorn hen from.Flock No. 1. Serum agglutinated standard
antigen in routine test 4+ in both the 1-50 and l-lOO dilutions at 24
hours. Killed 6 weeks after test.
Examination shows bird to be well fleshed. The shanks, beak, and

vent are yellow and the bird is apparently not in lay.

gecropsy'Findiggg=

The gross dimensionsof the ovarian mass in situ are 6 c.m. by

4 c.m. There are four pedunculated ova of irregular outline and angular

~86-

 

contour ranging in size from 1.0 c.m. by 8 m.m. by 5 m.m. to 1.5 c.m. by

l c.m. by 8 m.m. of a mottled greenish-yellow to brown color. All are in-
jected and petechiated and have a leathery capsule (caseation necrosis).
There are three ova 4 m.m. by 3 m.m. by 2 m.m. of angular outline, and
green to brown color attached by pedicels 5 m.m. to 1 c.m. in length, which
have a leathery capsule and whose contents are caseous. There, are six ova
spherical in shape ranging in diameter fron 5 m.m. to 8 m.m. and numerous
small ovales from pin head size to 3 m.m. in diameter of a pale yellow to
bleached color.

Serum Titre:

4+ in 1-640 and 2+ in 1-1280 dilutions at 48 hours.
Bacteriological Findings:

Salmonella pullorum was isolated from the necrotic ova. This
strain is agglutinated by positive sera 4+ in the 1-320 dilution. Acid
and gas are produced in Dextrose; acid only on Arabinose, Galactose,
Levuloee, Mannose, and Rhamnose; neither acid nor gas on Mannitol, Xylose,

Dulcitol, Lactose, Maltese, and Sucrose.

CASE NO. 15

History:
White Leghorn hen from Flock No. 1. Serm clouded standard

antigen on routine test in both the 1-50 and l-lOO dilutions. Killed
8 weeks after test.

Examination shows bird well fleshed, and apparently in lay
judging from external evidences.
Necropsy Find_igg_s_:

There are five normally maturing ova of a golden yellow color

whose diameters range tron l. c.m. to 3.3 c.m. There is one ovum which

1“.
we .
.‘F
.tla.
.15... _

”0' a

is irregularly ovoid and whose dimensions are 2 c.m. by l c.m. by 8 m.m.
which has a roughened capsule and is dark brown in color. The contents
are fluid, and because cultures remained sterile, this may be retrogressing
follicle. Remainder of ovarian mass present no striking features. Other
organs show no macroscopic evidence of disease.
Serum Titre:

Serum at necropsy failed to cloud or agglutinate standard antigen.
l§aoteriological Findings:

All cultures remained sterile.

CASE NO. 16
History:

‘White'fiyandctte hen from.Flcck No. 3. Serum clouded standard
antigen in both 1-50 and 1-100 dilutions on routine test. Killed 10 weeks
after test.

Examination shows bird in lay as judged by bleached appearance
of shanks, beak, and vent.

Necrcpgy‘Findingg:

The abdominal cavity contains two cysts in adjacent parts of the
mesentery. These cysts are ovoid in outline and measure 5.5 c.m. by 4 c.m.
by 2 c.m., and 7.5 c.m. by 5 c.m. by 3 c.m. respectively and are separated
from each other by a fold of mesentery 4 c.m. in length. The cystic fluid
is slightly turbid and contains small gray floccules.

There are three normally maturing ova which are spherical in
shape and yellow in color. Their diameters are 1.5, 1.8, and 2.2 c.m.
There is one ovum 5 m.m. by’4 m.m. of a dark brown color and has a roughen-
ed contracted capsule. Remainder of ovarian mass presents the normal
appearance of numerous ovules from microscopic to small pea size which

are of a bleached to pale straw color.

-88-

Serum Titre:

 

Serum caused a partial agglutination in the 1-20 dilution, but
failed to cloud standard antigen.
Bacteriological Findings:

microscopic examination of the cystic fluid and the floccules
for encysted parasites were negative. The floccules appear to be aggrega-
tions of desquamated mesothelium. Cultures of this fluid as well as

cultures of the ova remained sterile.

CASE NO. 17
Histcsy:

White Leghorn hen from.Flock No. 1. Serum agglutinated standard
antigen in routine test to 4+ in both the 1-50 and l-lOO dilutions in 12
hours. Killed 8 weeks after test.

Examination shows bird to be well fleshed and apparently in lay.
Necropsy'Findingg:

The ovarian mass shows four normally maturing ova of a golden
yellow color and ranging in size from the smallest with a diameter of 1.5
c.m. to the largest with a diameter of 3.5 c.m. A.vesicle containing
a clear fluid and measuring 2 c.m. by 1.2 c.m. by 1 com. is attached to
the anterior border of the ovarian mass.

There are five pedunculated encapsulated ova which show casea-
tion necrosis (See Plate VIII). Two of these are separatedfrcm the ovarian
mass by pedicels 3.5 c.m. in length. The other three are embedded in the
mesentery of the abdominal cavity 5 c.m. from the lower border of the
ovary. All have leathery capsules, and are resistant to pressure. There
is one follicle measuring 2 by 1.5 c.m. which is hemorrhagic and undergoing

liquefaction necrosis. Remainder of ovarian mass presents the normal pic-

-89-

" “‘2“ “‘

‘-——

 

 

: schl\\\n 9
s. '

 

 

PLATE VIII

Case No. 17. Note encapsulated ova suspended in the
band of mesentery on the right, and the pedunculate ova
on the left, which showed caseation necrosis. An egg

is in the uterus or shell gland. S. pullorum was isolated.
(Degree of enlargement 2/3 x).

-90-

ture of immature ova in various stages of development. An egg is in posi-
tien in the uterus.
Serum Titre:
4+ in 1-320 and 2+ in 1-640 at 48 hours.
Bacteriological Findings:

Salmonella pullorum was isolated from the necrotic ova. This

 

strain is agglutinated by positive sera 4+ in the 1-1280 dilution. Acid
and gas are produced in Arabinose, Dextrose, Galactese, and Manncse; acid
only in Levuloee, Mannitel, and Rhamnose; neither acid nor gas in Xylese,
Dulcitol, Lactose, Maltese, and Sucrose.

Staphylococcus aureus and Salmonellagpullorum were isolated

 

 

from the ovum undergoing liquefaction necrosis.

CASE NO. 18
Histogy:
White Wyandotte pullet from Fleck No. 3. Serum clouded at 12
hour reading of agglutination test. Read as 4+ agglutination at 24
hours. Killed 14 weeks after test.

Necropsy;Finding_:

 

General: There is a fibrinous exudate on the mesentery and
peritoneum, and many adhesions of variable extent are present between
folds of the mesentery and the peritoneum. The liver shows fatty degenera-
tion but is not greatly enlarged; it has two gray foci on one of the lebes
which are cultured. The heart shows fatty degeneration.

Ovarian Mass: There are seven apparently normally maturing ova

 

of a light golden yellow color ranging in diameter from 1 c.m. to 3.5 c.m.

The enveloping capsules of the largest two are petechiated. One ovum which

-91-

is angularly ovoid and measures 1.5 by 1.3 c.m. has a capsule which is
greatly thickened and hemorrhagic. It contains a fluid of greenish color
with flakes of a brown color (liquefaction necrosis). Two other ova are
pedunculated, have leathery capsules which are injected and hemorrhagic,

and contain caseous masses with some fluid of a brownish color. There

are several ovules which are pedunculated and angularly ovoid with diameters
of 3 to 5 m.m. The contents of these are solid and of a mottled green and
brown color. The number of immature ovules is comparatively small. An

egg is in position in the albumen-secreting portion of the oviduct.

Serum Titre:

 

4+ in 1-2560 and 2+ in 1—5120 dilutions at 48 hours.
Bacteriological Findiogg:

Salmonella_pullorum was isolated from the foci in the liver as

 

well as from the necrotic ova. This strain is agglutinated by positive
sera 4+ in 1—320 and 2+ in 1-640 dilutions. Acid and gas are produced in
Dextrose, Galactese, and hannose; acid only in Arabinose, Levuloee, and
Rhamnose; neither acid nor gas in Mannitol, Xylese, Dulcitol, Lactose,

Maltese, and Sucrose.

CASE NO. 19
Histogy:
White Leghorn hen from Flock P. Serum agglutinated standard
antigen on routine test 4+ in the 1-50 dilution at 24 hours. Killed 1
week after test.

Necropsy Findings:

 

General: There is a quantity of peritoneal exudate of a brownish
color. The folds of mesentery bear a number of areas of induration of

limited extent. There are reddish brown spots and areas of congestion

on the intestine. The liver is somewhat enlarged, has hemorrhagic areas,
and several minute grayish foci, but is not friable. Heart appears to be
normal.

Ovarian Mass: One ovum measuring 3.8 by 3.6 by 1.5 c.m. is

 

pedunculate, is constricted by the enveloping capsule into lobes, and is
of a mottled yellow and brown color. Contents are fluid and of a greenish
yellow color with brown floccules (liquefaction necrosis). There are two
pedunculate ova angularly ovoid in outline whose capsules are roughened
and somewhat thickened of a mottled yellow and brown color. These measure
2.5 c.m. in diameter, and their fluid contents contain lumps of a caseous
character. Another ovum of similar dimensions has a capsule which is
hemorrhagic throughout its extent save at the stigmen. One ovum measuring
1 c.m. in diameter is embedded in a fold of the mesentery acid attached

to the ovary by a slender pedicel. The capsule is leathery and its con-
tents are caseous, with no fluid (caseation necrosis). Numerous pedunculate
angularly spherical ovules, 3 to 5 m.m. in diameter with solid contents of
a mottled color are found among the immature ova.

Serum Titre:

 

4+ in the 1-320 and 2+ in the 1-640 dilutions at 48 hours.
Bacteriological Findings:

Salmonella_pullerum was isolated from the necrotic ova. Cultures

 

from the foci in the liver remained sterile. This strain is agglutinated

by positive sera to 4+ and 2+ in the 1-640 and 1-1280 dilutions respective-

ly. ‘Acid and gas are produced in Arabinose, Dextrose, Galactose, and Levup
W}

lose; and,only in Mannitcl, nannose, and Rhamnose; neither acid nor gas in

Xylcse, Dulcitol, Lactose, maltose, and Sucrose.

 

n,
—S.

GENERAL SUMMARY AND RECAPITULATION OF CONCLUSIONS

In an investigation in which the blood serum of 3,400 chickens
became available, studies were undertaken to determine the cause of, and
a means of preventing that non-specific precipitation known as the cloudy
reaction. Corrollary bacteriological and pathological studies were con-
ducted on selected cases. The means of procedure and the data secured

are presented. The conclusions are here briefly summarized.

l. The clouding reaction appears earlier than the agglutina-
tion reaction and affords no selection of time when clouding gires less
interference with a proper reading of the test. Prediction of clouding
by inspection of the condition of the serum is not possible. Clouding
'was encountered in greater frequency with the serum of females than of
males. The incidence of clouding is variable among different flocks,
and the work here presented does not indicate that breed is an important
influence.

2. Fat is present in the cloudy precipitates, and also is
present in greater quantities in sera which produce the cloudy reaction
than in those which do not; this is suggestive that fats are responsible
for cloudy reactions.

3. The serum probably is the principal source of the precipi-
tate in the cloudy reaction.

4. The method of drawing the'blccd samples, the variation of
time and temperature of incubation combined with refrigeration, and the
omission of phenol from the antigen do not afford a means of inhibiting
clouding. The addition of colloids to, increasing the viscosity of, and
the addition of a fat solvent, acetone, to the antigen do not prevent non-

specific precipitation.

The increase of the electrolyte content of the antigen by the
addition of 5% sodium chloride is a satisfactory means of lessening cloudy
interference, and appears to cause less disturbance of the antigenic sus-
pension than the addition of sodium hydroxide for the same purpose.

5. The diagnoses made as to birds giving specific agglutination
reactions, and these giving cloudy reactions appear to hare been accurate
by bacteriological and pathological confirmation; Salmonella_pullorum was
isolated from the positive reactors, but not from cloudy reactors in the
cases studied. Further, incubation at 37° C for at least 48 hours is
required to reveal all reactors by the agglutination method. There is
a variability in the fermentation of carbohydrates by recently isolated

strains of S.4pullorum, though these uniformly ferment Dextrose, but not

 

‘Dulcitol, Lactose, Maltese, and Sucrose.

 

 

BIBLIOGRAPHY

1. Mathews, F. P., Jour. of Immunology, 1926, Vol. II, pp. 499-504.

2.

3.

4.
5.
6.

7.

8.
9.

10.

11.

12.

13.
14.

15.

16.

17.

18.

19.
20.

21.

Proceedings of Thirteenth Annual Meeting, U.S. L.S.S. Ass'n, in Jour.
Of Me Vet. Med. ABB'H’ 1927’ V01. 70, pp 922-9250

‘Warner, D. B., and Edmond, H. D., Jour. Biol. Chem., 1917, Vol. 31,
pp. 281-294e

Hitchner, E. R., Jour. A.V.M.A., 1923, Vol. 63, pp 759-763.

Stafseth, H. J., Mich. Expt. Sta. Report, 1925, p. 196.

Stafseth, H. J., Mich. Expt. Sta. Report, 1924, pp. 186-187.

Stafseth, H. J., See No. 5.

Beaudettq,F. R., N. J. Sta., Hints to Poultrymen, Vol. 12, No. 9, p. 1.
Neumann, A., Cbl. f. Physiol., 1907, Vol. 21, p. 102. ‘
Oshima, T., Cbl. f. Physiol., 1907, Vol. 21, p. 297.

Stitt, E. B., Practical Bacteriology, Blood Werk and Animal Parasitology,
7th Edition, Blackisten, New York, p. 571.

Neisser, B., and Braeuning, H., Zts. f. expt. Path. und Therap., 1907,
Vol. 4, p. 747.

Leathe, J. B., and Raper, H. 8., ”The Fate" a.menograph on Biochemistry,
1925, Longmans, Green and Co., London and New York. See Chapter VI

for a full discussion.

Hadsen, et a1, Jour. of Expt. Med., 1906, Vol. 8, p. 337.

Rebraisser, Ra Ea, Jour. As Va He Am, 1926, VOIe 68, pp. 603-621e

Barton, E. F. The Physical Properties of Colloidal Solutions, New York,
2nd Edition, 1921.

Hecht, Hugo, Zts. r. Immunitat., 1923, Vol. 36, p. 321.

Northrop, J. H., and DeKruif, P. H., Jour. of Gen'l Physiol., 1922,
Vol. 4, pp. 662-667.

Huddleson, I. F., and Carlson, E. B., Jour. A. V. M. Am, 1926, Vol. 70,
pp. 229-233e

Runnells, et a1, Jour. A. V. M. A., 1927, Vol. 70, pp 660-662.
Hay, H. G., and Goodner, K., Jeur. of Bact., 1927, Vol. 13, pp. 129-146.

Beaudette, F. R., Jour. A. V. M. A., 1923, Vol. 64, pp. 225-227.

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