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A SUI-TV EY OF THE INCIDENCE
OF SEROLOGICAL VARIANTS
OF SALMONELILAPU LLORUM

IN MICHIGAN

[WM

b4
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TH

.0)

T H E S I S
FORTHE DEG-FEE OFM. S.
MICHIGAN STATE COLLEGE
E'AMES A. BIV‘INS

I947

 

This is to certify that the

thesis entitled

"A Survey of the Incidence of Serological
Variants of Salmonella Pullorum in
Michigan"

presented by

James A. Bivins

has been accepted towards fulfillment
of the requirements for

I. S. degree in Bacteriology

1//W

’ / Major prollessor

Dam December 18, 1947

 

 

 

 

PLACE IN RETURN BOX to remove [hlS c
TO AVOID FINES return on
MAY BE RECALLED with earhe

heckout from your record.
or before date due.
r due date if requested.

a ,

A SURVEY OF THE INCIDENCE OF SEROLOGICAL VARIANTS
OF SALMONELLA PULLORUM IN MICHIGAN

BY

JAMES A. gyms

A THESIS
Submitted to the School of Graduate Studies of
Michigan State College of Agriculture and
Applied Science in partial fulfillment
of the requirements for the

degree of

MASTER OF SCIENCE

Department of Bacteriology and PUDlic Health
Year 1947

THESE

Il-ETRODUCTI on

When discussing pullorum disease eradication with
hatcherymen, the diagnostician is often asked why repeated
blood-testing of breeder flocks has failed to bring about re-
duction or elimination of the disease in chicks. Sometimes
one can find management practices which are at fault. In
other cases, however, one is at a loss to explain this failure
to his own satisfaction; much less to the satisfaction of
the hatcheryman. The reports of various Canadian workers
concerning the presence of so-called "variant" strains of
Salmonella_pullorum have offered another possible explana-
tion. It was felt that a survey to determine the incidence
of "variant" strains of g; pullorum among cultures isolated
from fowl presented for diagnosis at the Poultry Clinic
might aid in the evaluation of the importance of this aspect

of the Michigan pullorum disease control plan.

REVIEW OF LITERATURE

Younie (10) states that in 1939, a few outbreaks
of pullorum disease occured in chicks hatched from breeders
. which were negative to pullorum disease tube tests. In 1940,
an increase in the number of such outbreaks was noticed. A
retest of the breeders of one breed revealed only 11 reactors.
Losses continued to the end of the hatching season. Neither
the post-mortem appearance of affected chicks nor the cul-
tural characteristics of the organisms isolated from them
differed from the usual findings in pullorum disease. Cer-
tain serological differences were seen. slide agglutination
tests showed differences in rapidity and type of agglutination.
In tube agglutination tests, antiserum of the pullorum variant
did not react with Eastern Conference strains 19 and 20 and
gave only a 1+ reaction in 1/50 dilution with strain 17 while
-three variant strains gave complete agglutination in a 1/800
dilution.

YOunie reported further study in 1942 (11). A
flock of 94 pullets was tested for pullorum disease in the
'fall. Three percent were found to be reactors. A retest
made six weeks later showed no new reactors. On July 5th
of the following year, the flock was tested with.antigens
made from the three conference strains, five variant strains,
with three commercial antigens, and with.one commercial whole-

blcod antigen. Serum from three birds reacted with the con-

- 2 -

ference strains' anti ens and commercial antigens while 64
sera reacted with the variant strains' antigens. Cultures
of §; pullorum were obtained from 65$ of 90 birds autopsied.
Additional history supplied after the study was begun indi-
cated that the variant infection may have started in the
spring preceding the July test. A flock of baby chicks pur-
chased at that time suffered a 40% mortality from pullorum
disease caused by a variant strain.

Attempts to verify Younie's reports and to find
an explanation for them have been made. Glover and Connell (5)
used 47 cultures of g; pullorum obtained from consignments
sent to the diagnostic laboratory. Agglutinin absorption
tests showed that the strains fell into at least two groups-—
"normal" and "variant". Low titer sera from experimentally
infected birds agglutinated the homologous antigen but did
not agglutinate the heterologous type. "Consequently," they
state, "infected birds carrying a low serum titer are in
many instances not detected by the standard agglutination
test." Byrne (1) used ten strains of g; pullorum and their
respective rabbit antisera in his studies. He found cross
agglutination between variant and standard strains but he
points out that the titers of these sera were higher than
might be expected to occur in fowl naturally infected with
the chronic form of pullorum disease. Agglutinin absorption
studies showed a distinct division into standard and atypical

or variant strains.

Wright (9) reported that, in over 7000 blood

Ha

samples tested by the tube agglutina tion m; th 00 with ant
gen of ooth conference strains and variant strains, 55.c«
were positive with variant antigen but only 1.4fl were posi-
tive with conference strain anti en. ii: i113 conference and
variant strain 5 in"aliquot” p1 oportion yielded an antigen
intermediate in results between its two components.

Gwatkin and fond (8) infected 350 chicks with five
var riant strains of 2; nullorum. Of 294 which were tested

for agglutinins at five to six weeks of age, 191 were p081-
tive with variant anti en but only 20 with r gular type
antigen. fherefore, about 59» of the reactors would have

b en missed by the regular antigen if positive and suspicious
reactions were grouped. A second test was made between 80
and 102 days of age. Regular (or conference strains) anti-
gens failed to detect 65.3; of the oil ds whose sera were
either positive or suspicious with variant antigen. In 815
tests, only we (0.25;) kn wn negative birds reacted with
variant antigen.

This study was continued by Gwatkin (5). At five
and a half months of age, 19.3% of the chickens positive
with variant anti ;en were ne ative with re gular antige en.

At this time, tr e fowl were divided into two flocks; one
positive and one negative. The negative group (110 birds)
was tested at seven and a half months; ten reactors to
variant antigen were found. Five of these birds did not

react to regular antigen; however, 5. pullorum was recovered

-4-

from seven. At nine months, two reac ed with variant anti3en

and one of t31ese with the re ular antigen. it ten months no

x...‘

q

reactors were found with either anti3en in either the plate
or tube tests. At one year of age, one hen reacted with

variant antigen but was no ative to the recular anti e

{3

L
M.

s. pullorum was not recovered. Thre settings of eggs from

each of the positive and no :ve flocks were made. The

{D
Cl

mortalities for each of the setth1;s from t11e negative group

were 12% , lBfl , and 101; S. pullorum was not recovered from

‘W

L1

any of UflCSCo fhe percent mortali tie es in oiaicks hatched from

.‘I

f‘.

the variant positive group were 84, 42, and 5. The pullorum

C

organism was recovered from 100 ,eo; and W1 , res;. actively.
Gwatkin states, [323] "It appears that variant infection was
controlled by the same methods employed in dealing with

egular ty infection with the very important difference
that a variant antigen was on: loy

Gwatkin resorted another study of serological

reactions of variant g; pullorum infections in 194d (7).
Sixty birds which had been infected by mouth as chicks with
variant strains of g; pullorum were killed or died between
the 6th and 17th mont:1s after infection ani bacteriolOf ica al
examinations we re made on these birds. All were positive in
tube ag3luti nation tests with variant antigens. Eight of
the 60 had the same serum titer with both antigens; the re-
mainder had hi3her titers with variant than with regular

antigens. Thirty-two were negative with regular antigen in

- 5 -

p;

ilutions as low as 1/10. The organism was recovered from

5 of these. Bourteen of t31e 52 reacted only in a 1/2

{\T)

dilution with variant antigen and the organism was re covered
from nine of these. Seventeen other birds of the 60 reacted
with regular antigen only in a 1/10 or a 1/20 dilution and

the organism was recovered from 15. with advancine

K.)

a e,
fewer birds were missed by the regular antigen but during
the age when they would iormally have been tested, many would
have been missed by regular antigen only.

Some other observations which Cvatkin made during
the course of his studies are mported (7). Parenteral in-

"ive clear cut

fection with vai iant t’pe strains did not 0
results and serum had to be collected early to detect dif-
ferences in agglutinins. Absorption experiments showed that
re ular a bglutinins were more easil1 y removed by either
regular or variant strains than were variant a 3f;lutinins.

In some cases, however, massive treatment with either reg ular
or variant type organisms removed both types of agglutinins.
That an antigenic dif elence between variant and regular
types of g; nullorum exists was shown by cross agglutination

tests with a strain of Proteus sp. isolated from the ovary

 

.l

of a hen whose serum agglutinated variant antigen. Eighteen

variant-strains were agglutinated in fairly high titer

(1/160 to 1/640) in Proteus antiserum. Thirty regular

strains were arvlutinated only in a 1/10 or a 1/20 dilution.

Qt.)

Gwatkin (6) tested 746 birds from variant infected
flocks and 645 from regular type infected flocks using both
tube and plate tests and with variant, regular, and mixed
antigens. whole blood antigens gave an agreement of 99.4%
between variant and mixed antigens and 96.8% between regular
and mixed antigens. In the first tube test comparison,
positive and suspicious reactions were grouped. nixed anti-
gen detected 98.65 of reacting birds detected by variant
antigen and 98.4p of regular antigen reactors. Regular anti-
gen detected only 65.73 of the variant reactors. In the
second tube test comparison, agreement between Variant and
mixed antigens and regular and mixed antigens was 97.5»
and 92.8p respectively, when tested at 1/50 dilution. when
tested at 1/25 serum dilution, the agreement was 94.95 and
90.2fl,respectively.

Finally, a detailed antigenic analysis has been
made by Edwards and Lruner (2). The authors state that
the antigenic formula of §; pullorum is IX, XIIl’ ikII2;]
XIIS. In normal cultures, the XI12 factor is variable, and
forms containing a large amount or a negligible amount of
XIIzcan be isolated from the same normal strain. It is
possible for cultures to become fairly well stabilized in
either form, thus giving rise to the so-called "standard"
(St.) strains and "variant" (X) strains._ The standard strains
contain only a small amount of X112 but the variant strains

contain a large amount.

The reactions in §; ,aratypgiié_var. durazzo serum
(II, X111, X115) and g; reading serum (IV, XIII, XIIB) were
particularly revealing. The Proteus serum of Gwatkin Save
results comparable to those obtained with S; reading serum.
The Proteus culture used to prepare the serum contains all
or a portion of XIIZ. It was evident that §t_pullorum St.
had a very weak XII2 content whereas S; pullorum X had a

very strong XII component.

2
When S.TYEhi 0901 (IX, X111, X112, X113) 03? 3: p111-

lorum X serum was absorbed by §:_ty;hi T 2 (IX, XIIl, X115)

 

a strong residue of agglutinins remained for s. pullorum X,
S. typhi 0901 and S. reading. This residue represented the
antigen XIIO. When the same sera were absorbed with S.

‘J —

pullorum (standard), a much weaker residue of agglutinins

 

was left for S. pullorum X, S. typhi 0901 and S. reading.

 

This indicated that while the XIIZ component was not suf-

ficiently developed in S. pullorum St. to cause evident

 

agglutination, a sufficient amount was present to absorb
most of the XII2 agglutinins. This conclusion was sup-

ported by absorption of S; pullorum St. serum with S. typhi

 

T 2. A residue of XII2 agglutinin was left in the serum

which agglutinated S. pullorum X, S. typhi 0901 and S. reading.

 

Apparently, while S. pullorum St. did not contain sufficient

X112 antigen to be agglutinated, it did contain enough to

produce a low titer of X112 agglutinins.

When durazzo serum was absorbed by s. readinc

it still agglutinated é; pullorum X in a low dilution,

indicating that the variant strains contained a small

amount of XI 3 antigen. S. reading serum absorbed with

durazzo no longer agglutinated S. pullorum St. indicating

 

 

that the standard strain did not contain sufficient X112

antigen to cause it to agglutinate in X'IO serum.
9

The X1121 and X112r+ forms were easily dis-
tinguished in the slide agglutination tests using S; reading
serum absorbed by durazzo (X112) and durazzo serum absorbed
with S; reading (X115). The ++ forms agglutinated rapidly
in K112 serum and very slowly r not at all in KITS serum.
In the 3 forms, the behavior was just the reverse.

An attempt to differentiate the wo forms of
.§; pullorum by using a phage (4) isolated from organs of
chicks naturally infected with a regular strain of_§;
pullorum showed no significant difference in activity for
any of six regular or five variant strains tested.

A few statements concerning the relative incidence
of variant infection occur in the literature. The outbreaks
of pullorum disease in chicks which Younie reports (10,11)
were primarily of the variant type. Glover and Connell (5)
typed 161 strains of g; pullorum isolated from post-mortem
material and found 110 variant strains, 49 normal (or stan-
dard or regular) and 2 which agglutinated with both sera
equally well. deference has already been made to Wright's
(9) series of agglutination.tests.

-9-

‘ r a. -‘ if") A“. L n F T "i' "’ -V ‘3..'].r_ff\fl\ '1
l'JLrlu-LIn '0 A3...) irAJ;.L-\J.1i/:

Two hundred and two cultures of Salmonella pul-

 

lorum representing 199 consignments were isolated from
chickens and turkeys of various ages presented over a
period extending from Iovember l, 945 to September 1, 1946.
In many instances, the consignments were shipped to the
laboratory. Often it was not possible to learn the name
of the hatchery from which the chicks were purchased. hence,
it is not known how many different hatcheries are repre-
sented nor how many of the strains collected originated
from the same hatchery's stock.

Isolations were made by streaking material from

.,

birds suspected of having pullorum c

4‘.

isease on bifco "SS."
agar plates. After 24 hours incubation, colonies having
the gross appearance Of.§; pullorum were subcultured and
identified. Usually after identification, one culture,
chosen at random from eac consignment, was pieced in stock.
Stock cultures were maintained by stab seeding semi-solid
agar containing 0.5} beef extract and 0.5% proteose-peptono.
These were incubated 48 hours, sealed with paraffin, and
stored in a 56° to 40° F. refrigerator. The stock cultures
were transferred at 4 to 6 week intervals.

An antigen was prepared from each strain in the
stock culture collection by seeding about 5 cc. of broth

with a loopful of the stock culture. The broth tubes were

_ yo -

incubated for 8 hours at 57° C. on a mechanical horizontal

fl

i'his procedure "ave rather uniformly good growth.

C)“

shaker .
Each broth culture was then used to seed agar slants gon-
finned in 6 or 8 ounce screw-cap prescription bottles. The
slants were incubated in a vertical position for 40 hours.
After incubation, the surplus broth inoculum was aspirated
and discarded. The bacterial growth was washed from each
slant using about 5 cc. of have iological saline per slant.
This yielded a very dense suspension of live orQa ni ans.

Gwatkin (7) resorted that antiserum :rep ared Wltfl

a st; ain of Proteus obtained from the ovary of a hen ag—
glutinated variant strains of S. pullorum in hi ;h dilution
but did not a”i:lut inate standard strains beyond 1/20. He

k."«

kindly supplied a transplant of thi

U)

Iroteus strain. An
antigen was prepared from it by boiling the growth washed
off 3” a 24 hour a Qar slant cultu e. A rabbit was hyper-
immunized by 4 intravenous injections of 0.5 ml. each at 4
day intervals. Another rabbit was similarly in munized with

Salmonella n ratyghi A vari miy durazzo. A third rabbit was

 

imxn mzized with é; pullorum, Sastern Conference strain 19.
Four days after the last injection of antigen, each rabiit
was bled by cardiac puncture and the serum was harvested.

V The 202 recently isolated strains 0f.§; pullorum
and 6 strains maintained in the laboratory for several years

were typed b’ olacin” anoreximatelv 0.02 ml. of each of the
J. .L x.) 1.1. d

hree se a on a glass plate and mixing with each serum a

_ 11 _

sirH1Llar quantitv of antigen. If aQQlutination was not in-

mediate, the plate was "sit ; rotated. Ehe intensitv of ti

6

reaction was not classified except in these strains which
were positive to Proteus antiserum. with the puller ur
antiserum used, all strains, xcept number 600, gave a defi-

1

nite positive reaction. Strain number 800 was classified

as 2‘ or partial. The paratyphi A antiserum rarely gave

more than 2* reaction with any of the pullorum strains.
”ince most of the typinQ of pullorum strains re-

ported prior to the time of this study had been done with

the tube a'

QQlutination test, it was desired to check the
results of the serum plate test with those of the tube test.
Accordiany, antiQens traoa;et from 54 strains were each
diluted to a hefarland neohclometei LurDluiuJ of l and com-

bined with Eroteus antiserum and with S._paratyphi Av ariety

 

durazzo antiserum in serum dilutions of l/2d, 1/63, 1/160
and 1/320. These were incubated 20 hours at 50° C. in a

water bath and 4 hours at room temperature.

q _-

The results of the serum—plate typinQ o; loo
strain s are recorded in Table l A. The results of the
typing of the ”mi r 52 cultures, 8X31 ns d by both the Serum-
plate test an' the tube test, are recorded in Table l B.

Qhe results of all of the serum- plate sets are suimari zed

in Table 2. 01 the 51 strains (15; of the total) w.h:ich a;

glutisza ted with Eroteus antiserum, 29 agglutinated quickly

J

and distinctly. The cell aggregates were, however, smaller

-12..

Table 1 A.

Serum Plate Agglutination Results

 

Consignment Antiserum

 

Number Proteus Para A Eullorum

 

 

917 .quwmhmflaluunu-WMnmmmmr-.—.— .- A.. w- .

45 - 591
595
605
507
627
628
629
651
656 a
657
658
640
644
646
650
655
657
658
654
681
686
688
690
691
695
694
695
698
709 .
711
712
715 i
714
715
716
719
721
722
725
728
752

A.

.)
#-

 

-U 9‘51“.“ “K“ ““ ‘

H
+

(‘1

ll
f++++t+++f+~t+ +++++++++++~+++++~t++++++++++1-

')

fl -:U-o-,. r-.-.fim|v’".,'“fl_ ml.” ‘-,--1‘ gag-‘4. K .1

Illlllllllitl

v w . Unwrzl‘ l‘? f Wt a‘V’VV‘Hfi-i-nrvfiwnmvufiu 'U-Vr I‘ve: www.min‘x—‘V:

 

 

++i~+++++1~++++++++++n~+++ 1+ r++++++l++l++l1++++

0‘ on... . "-63.1.

.. 15'...

 

Table l A. (continued)

 

Consignment Antiserum

 

Number ioteus *THra A Pullorum

 

 

46 - 756
758
759
741
742
745
745
746
748
755
754
756
757
759
760
761
765
766
768
769
770
771
772
778
781
782
785
784
786
788
810
811
815
824 h
865 ‘
876 1
880 4
884 1
885
886
887
888
892

b)
ll‘I-llll

t3

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l\‘)

V 1"}...—

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l—J

AA.._- m—_

,p
+‘f1“f‘f1'+-t1H+-t4-+ub1“+-r1-+-riu+-fi“+'r1-+-ri-+-91'+'r1-+‘ffi'+-b1-+

N l\
+

t I II I II + I

C»)

 

7;“ I‘VI‘IJr“ .I-

 

1+f+++4l4+I+4-H-I-4+4é+~h+++++l+++4+++1l++fl++++++

rn.‘ Ln. “A“: s' at..-)
m. union-rum.

 

 

 

Table l A.

(continued)

 

Consignment
Number

Antisor

um

 

Proteus

Esra A

PuIIOrum

 

 

46 - 895
894
895
887
898
8‘39
900
902
904
906
907
908
909
910
912
915
917
920
922
950
951
955
954
957
958
959
949
955
958
961
962
965
967
968
971
975
988
995
996

1005
1006
1007

 

(\3
llllllll+lllllllllll

2

 

I +

.4!- -..u-I.—.v5vm

- 15 -

-+‘OI‘+-rfi“+‘rfi“rfi*l-r1*+-#fl~f'91"T-r1‘r—t1~f‘l1‘fflr+ +1~+-r1~+-t1~+-r

Ah“; .1.-

 

 

(A
fifl¢++1+++++¢+++++++++++¢++++++++++++++++++

OJ

 

‘7 was. 7“"

Table l A. (continued)

 

Consignment
Number

Antiserum

 

‘Proteus Eara A

Pullorum

 

46 - 1040
1042
1069
1071
1079

A - 4

6 - 15

57
48
50
64
70
79
87

105

105

106

117

122

164

165

170

166

196

179

19

1‘5 1 7

M 19

M - 20

Variant 201
Proteus

“3

P
P

m
lllll+lllll

10
+

N!
l1-

(‘3
+

I
II +«94~+-r4-+~r4-+«I|-+~rl +«Ir++-r4-+m+r++-+4++u+

wsw
+.+

 

“ts

#u #3

(,0

m
+++++4+++++++++4+++++++++++++++

 

 

 

 

~ 16 _

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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1

Lose which formed with pullorum antiserum; nonce,

n.- ‘.-I I9 , 0‘ “I _:f\ . “ ' ."‘ .7 a L I 'L' _O ‘. ' f ~- .- . >-
LIL;G:,T Vial/‘63 CldSSIIl’dd as Olll‘) (41' I’GJCULUIISO -‘JH' Str'alfls’

although positive, were considerably slower in aeflutinat’

I "\ “
"' (IJC. *ifli.)

and the reactions were classifies
The tube agglutination tests and the serum-plate
agglutinaticn tests were in good a

actions did not occur in any of the tube agglutination

test dilutions of Proteus antiserum which were not also

 

positive to the serum-plate test. Only one strain, number
854, agglutinated in the serum-plate test but di‘ not ag—

clutinate in the tube test. E0 explanation for this is

C)

off red.

(L)

Summary of serum-plate tvpinz of 208 strains of S. Eullorum.

 

Proteus antiserum Number of Sgparatyuh .§ var. Humber of

J.

 

reaction Strains Jurazao anti—serum Strains
reaction

 

+
(D

14

[4'

 

 

 

175

4.

- 177 i 2

 

 

 

 

 

-19-

DISCUSSION

Edwards (2) has shown that the so—called variant

u.

strain of S. oullorum is a form in which the procuction of

large amounts of X112 antigen has become stabilized. The

"standard” strains of S. pullorum contain small amounts of

 

the X112 factor. Ee found that recently isolated strains,
considered to be the ”normal” form, gave rise to two forms
of colonies; those having much.and those having little
X112 factor. However, these forms when transferred twice
on agar again gave rise to both forms. In other words,
they did not stabilize in either form. N Inasmuch as the
antigens tested represented the growth of masses of colonies,
it is felt that only those strains which originally were
stabilized in the variant or X112 ++ form reacted with
Proteus antiserum. Variant forms probably did exist in
some of the other strains but were not detected because
the strains were not stabilized in that form.

The strains which had been maintained in stock
longest were examined for the presence of rough colonies
and were found to be quite smooth and entirely suitable

for this survey.

SULJI‘LARY

Two hundred and two strains of S. nullorum iso-
lated from consignments presented for diagnosis at the
Poultry Clinic and six laboratory strains were classified

as variant or standard tyre according to their reaction

with Proteus and with S. Earatyghi A var. Lurazzo anti-

 

sera. The serum-plate test was used. Thirty-one strains
(lSfi) of the variant type were found. The results of the
serum-plate test were in good agreement with the results
of the tube test. In one case ("A" strains) where the
flock history was known, and, despite repeated testing of
breeders, pullorum disease persisted, it was found that

4 of the 5 strains examined were of the variant type.

- «7/hv-‘H.—. ""v 7wv‘-'ffl
Aplu‘i’ ‘x/‘ i iJ-J'AJJJ La: -4$‘—J.‘J.‘u J.

The writer wishes to thank Dr. Ronald Cwatkin,

DiViSiun Of Animal Path01053: Science Se

e

vice, Dominion
Department of Agriculture, Hull, 5uebec,for suggestions

and advice and ior cultures of variant S. pullorum and

*r!

 

roteus spp. Dr. I. F. huddleson demonstrated his technique
for distinguishing smooth and rough colony forms. Dr. Hm.

Fervuson LlCthan state De artment of health Laboratories
L. ’ C.) ,

suonlied a transolant of S. -aratv hi A variet‘ciurazzo.
-4. do u‘ —

 

Finally, the invaluable ssistance of Lrs. Carl Eleil in

identifying and maintaining stock cultures of 5. rullorum

isolated from field cases is acknowledged. Without her

loyal efforts this study would not have been cossible.
J J l

BIBLIJGRAPHY

l. Byrne, J. L. Variant or Atypical Strains of Salmonella
pullorum: A Serological Study. Canadian Journal of
Comparative hedicine, Vol.7,No.8,(Au3ust,1945),pp.227-238.

 

2. Edwards, P. R. and D. W. Pruner. Form Variation in
Salmonella pullorum and Its Relation to X Strains.
The Cornell Veterinarian, Vol.36,No.4,(October, 1946),
pgogld'524o

 

5. Glover, J. S. and R. Connell. Studies in Pullorum Disease.
Report of the Ontario Veterinary College for 1942.
pp. 19-28. '

4. Gwatkin, R. Studies in Pullorum Disease. IV. The Effect
of BacteriOphage on Regular and Variant strains of S.
pullorum. Canadian Journal of Comparative hedicine,
Vol.9,No.2, (February,1945),pp.43-45.

5. Gwatkin, h. Studies in Pullobum Disease. V. efficiency
of Homologous and Heterologous Antigens in Detecting
Reacting Birds in a Variant-Infected Flock. Canadian
Journal of Comparative nedicine, Vol.9, No.7,(July,1945),
ppolUS-lglo

6. Gwatkin, R. Studies in Pullorum Disease. VI. Comparison
of hhole Blood and Tube Tests with Regular and Variant
Antigen and a Combination of the Two Antigens. Canadian
Journal of Comparative hedicine, Vol.9,No.6,(August,1945),
pp.216-225.

7. Gwatkin, R. Studies in Pullorum Disease. IX. Serological
Reactions of Regular and Variant Types of Salmonella
pullorum. Canadian Journal of Comparative hedicine,
Vol.10, No.9,(Sept mber, 1946),pp.254-267.

8. Gwatkin, R. and Eric w. Bond. Studies in Pullorum
Disease. 111. Serological Differences in Strains of
Salmonella pullorum. Canadian Journal of Public Health,
Vol.36, (April, 1945),pp.160—165.

9. firight, h. L. Pullorum-Like Variants and Non-specific
Reactors. Abstracted in Proceedings of 16th Annual
Conference of Laboratory Workers in Pullorum Disease
Control. (June 8 and 9, 1944),pp.13,l4. Storrs, Conn.

. 25'-

10.

ll.

Younie, A. R. Fowl Infection Like Pullorum Disease.
Canadian Journal of Comparative Ledicine, Vol.5, 10.6,
(June,194l),pp.le4-167.

Younie, A. R. Pullorum Like Infectian of howl. Canadian

Journal of Comp rative Ledicine, Vol.6,Ho.o,(June,1942),
p)? . 1.7.2-173 .

 

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