2

I

   

STUDIES ON BACILLARY WHITE DIARRHEA
OF THE DOMESTIC FOWL

THESIS FOR DEGREE OF M. §,
JAMES ALEXANDER BERRY

1917

 

 
Au. Siebert
Crates

~ plage aye

n-oIinders

ings. Mich.

 

THESIS
STUDIES Cif DACILLANY WiilTE DIARRHEA
Q

be

Tus DOMESTIC Civil.

THESIS

Subnitted to the Faculty of the lichigan
azricultural College in fartieal Dulfillment of the

requirements for the Degree of Master of Science.

James Alexander Berry.

June 1917.
THESIS
COWHIMNS,

Introduction.

Historical Resund.

Experiments.
Morphological and Cultural Characteristics.
Fermenting Power in Saccharine Broths. |
Thermal Death Point.
Resistance to Disinfectants.
Longevity on litter. |
The Agglutination Test.
The Complement Fixetion Teste
The Intradermal Test. |
The Infection or liature Howls.
Pathogenicity for Experimental Animals,
xesistance to Cooking. |

Additional Remarks.

Summary and Conclusions.

Acknowledgments.

references.

Platese

93910
STUDIES OW BacILLAxY WHITE DIARRHEA

OF THE DCRZSTIC PCL.
Introduction.

Domestic fowls of all tzpes have up to the present
received but comparatively scant notice from the scientific
world. The embryologist, it is true, has hed recourse to
the egg for olass-room work, for which purpose it is, indeed,
by its abundance and cheapness well adapted. But these very
qualities that recomend it to the exbryologist have been
largely responsible for the meagre attention bestowed by men
in other branches of science on the esg and tne hen that pro-
duces ite An individuel fowl, except in rare cases, is worth
but little, and the average farmer pays smell attention to its
10S8. Hence, until the eavent of the comsereial poultry plant,
the domestic fowl occupied a position in the economie world in
no wey comparable in importance with that occupied by such
animals as the horse, cow, or pige Lacking thus in strict
money value, the domestic fowl hes been studied more or less in
a purely scientific spirit. Treatises on its anatomy and
physiology are rare, and the treatment of its diseases is largely —
enpirical. Of late years, however, the great expansion of the
poultry industry nes drawn attention rather forcibly to poultry
diseases, and stimuleted investigation in veterinary and bacteri-
Ological circles.

While o1d diseases such as tuberculosis and roup must
be fought by the poultryman, others of leter origin must be con-

tended with es well. One or the most recent and cestriuctive
of these is bacillary white diarrhea, It is not too mich to
say thet this disease is appreciably cripplirc the poultry in-
dustry in not a few perts of the Unitec States. Not only is
the yearly money loss in young chicks great, but the destruction
of so meny well-bred birds hinders the development of strains
desirable for utility or show purposes,

Considerable investigations have been carried on con-
cerning bacillary white diarrhea, but there is room not only for
work on some hitherto untouched phases of the disease, but also
On corroboration of results alreacy published, It has, there-
fore, been deemed wise to cover some ground more or less thorough-
ly gone over already, and to conduct newer experiments as oppor-

tunity offered.

Historical Resumé.

In 1900, as a result of high mortality from an unzxnown
disease among very young chicks in certain Eestern sections,
Rettger (1) started investigations and isolated an organism with
which he was able to produce the diseese by artificial inocula-
tione Studies on furtner outbreaks in succeeding years con-
firmea his earlier observations, viz.--- that the septicemia is
due to a specific bacterium, usually affects chicks under four
days old, causing drooping, a whitish discharge from the vent,
loss of appetite, failure to grow, with death as a rule quickly
supervening; autopsy showing the liver with congested patches or
congested throughout; crop, lungs, kidney, and spleen apparently

normal, unabsorbed yolk usually presenting abnormal color or
' consistency. irom chicks dying of the diseese it is usually
eesy to isolate the casual organism, nemed by Rettger (2), in
1907, Bacterium pullorum. A valuable contribution to the sub-
ject wes meade shortly after by Rettger and Stoneburn (3) who es-
tablished the fact that mature hens harbor the organism and lay
infected eggs, the chicks from wnich, if they survive, in turn
become carriers os the disease.

Working independently of these two investigetors,
Jones (4). corroborated some of their work.  esults arrived at
by Gage (5) about the sane time were also corroborative in nature.

In 1912, Rettcer (6) secured some encoureging results
in the use of sour milk in lestenin,; the mortality among young
chicks exposed to infection.

With the knowleae thet the nucleus of thie diseese lies

n the ovaries of infected nens thet are to all az:peerance normal,

-

investisetors naturally sougnt to verfect a metnod of detecting
such carriers. Rettger (6) experimentcd with the egg test, as
also did Gece (7), but the results were not gratifying. Neither
was the agglutinetion test, first tried by Rettger (8), immediate-
ly successful. It was, however, later perfected by Jones (9).
The value of the test hes been well demonstrated, large numbers
of hens reacting to the tests of various investigators having
been found to exhibit typical ovarian symptoms of the disease on
post-mortem examination.

It is unfortunete, but understandable enough, that
practical poultrymen have failed to distinsuvish between becillary
White diarrhea, and more or less fatal ailments sinulatine, it is

true, bacillary white diarrhea, but usually originating in im-
proper feeding. serious losses among young chicks through
errors in feeding heve ceused rany poultrymen to ascribe bacillary
white diarrhea as the cause, end to svtspect healthy birds as
cnronico cerriers of the disease. Such mistakes have hindered
the cathering of reliable data on the extent of the cisease.
Loreover, such misconceptions are not confined to the ordinary
poultryman alone. For example, when Morse (10) ascribed Coccidium
tenellum es the cause of a diarrhetic outbreak which he investi-
geted, it was esswsed by Herzog (11) that this ovtbreak was ident-
ical with those epidemics studied by wettger. nince Morse af-
firmed thet the disease he investigated attacks chicks from two
to five weeks in age, the two outbreaks clearly must nave been
different in character. The term "bacillary white diarrhea"
which has come to be recardea by bacteriologists and veterinarians
as the result of Bact. pullorum infection and nothing else, is
accepted by others as coverins a wide range of intestinal troubles,
and its simplicity is more than counterbalanced by its lack of
specific meanins.

the disease in the course or some fifteen years has
made itself felt in probably every State (12), and has been ob-

,

served in imported birds as well (13).

Liorpnolozy ana Cultural Cheracteristics
or Bact. pullorum.

Six different strains were studied. the following
applies to all:-
Size: 1/2 micron by 21/2 microns on average.

a

Lotility: Uo true motility, but Brownien movement pronounced.
Air xequirements: Facultative anaerobe,.

Stains: Headily stained by usuel methods. Gram negative.
Not acid fast.

spores: lio spores have ever been observed,

Growth: Ager Slent,- most vicorous at 37°-'38°C. Delicate,
Shining, never spreading far from petn of inoculation; Will grow
about equally well on ordinery liver or glycerin evar; szgar be-
Ginning to dry out favored. On eg,ar pletes colonies very deli-
cate, round, trenslucent, sometimes difficult to detect,

Gelatin Stab: lo liquefaction; slight granuler
srowth elong inoculation line.
Broth: tiven turbidity in 24 hours. Sediment on
longer incubation.
Egg medium (Lubenan's -14-) Rather scent to mediun
erovth.
Indol production: Mone in Dunhem's peptone solution.

litrates: lot re.uced in nitrate peptone solution.

Fermenting power in suger broths: ‘The strains showed some

Slight differences, as indicated in the tables.

Dextrose Broth.

 

Strain. Gas in closed arm. Ratio of CO, to other Zasese.

 

Al Trace. lot tested.
3 _ , ; |
C Trace ilot tested.
W15Y 10% 122.3
~6=

liasltose Broth.

 

Strain. Gas in closed arn. Ratio of COp to other gases.

 

Al ~ -
AZ ~ -
C - a
W15Y - ~
RLY - -

 

pacehnarose Broth,

 

Strain. Gas in closed arm, ratio of CO> to other gases,

 

Al Trace. lot testec.
AZ _ -
C ~ ~
W15Y ~ -
Ri5Y - -

 

Lactose Broth.

 

Strain. Gas in closed arn. Ratio of COp to other gases.

 

Al 15% : 1:27
, - z
C 10% 1:3.0
WL5Y - _

RLSY Trace. lot tested,
Deternination of Thermal Death Point

of Bact. pullorun.

To determine the thermal deeth point of Bact. pullorum,
pieces of glass tubing were crewn out into cepilleries avout
10 ine to 12 ine in length. EHech ceapillery was flemed in the
middle until it separated, and then flamed where it merged into
the tube from which it wes drawn. Thus, two cepillary tubes
were Secured from one drewinz, each about six incnes long, with
@ bulging end and both ends sealed. The organisms to be tested
were in 24 hour broth culture. The method of filling the cap-
illaries wes to break oif the tip witn a heated file, flame two
or three times, and insert the tip in the broth culture after
heatin. the bulging end slightly. The aeajr contrecting as it
cooled in thc thick end resulted in Some of the broth culture
being forced into tne capillary. Every effort wes wade to
Secure cepilléries of the came Size and to crew the sare volume
of broth culture into each -- viz» 11/2 in. Each tube was
taken from the broth culture before the heatca air in the thick
end naa reacned its limit of contraction, so trat the lower end
of the column of broth culture eventually stood at least 1/2 in.
from the end oi the tube. The tip was then sealed by flaming
ana the tube laid in a water bath at the desired temperature,
the flame having been carerully regulated to maintain tret
temperature. At the end of ten .imtes the tube was quickly
withcrawn end immersed in cold 1:1000 HgClp for three minutes.
Handling with flamed forceps, it was then washed in sterile dis-

tilled water and inmersed in 95%0 alcohol for about ten Seconds.

On withdrawel from the alcohol it dried cuickly. The tip was
then broken witn a flaeiied file on sterilized filter paper and

the contents forecd into a tube of brotn by neatine the bulging
° . 2 ry ° - a |
end. The broth was then incubated at 57 ©. ana read at tne

end of 48 hours.

The first tube wes heeted at 40°C. for 10 mns., the
second at 42°C. for the seme time, and the remaining tubes
were heated in like :anner, each one two cezrees above the
precedinge There were 16 tubes in all, the hishest temperature
employed bein: 70°C. See table.

LW thermoneters were used in the water beth end the

teriperature wes not allowed to very morc than a frection of

v

one degree venti ,rede, while a tube wes being hected.
— 9

ray. sy: ‘NAaAt wT + KFC 7 he A I7 ~9979355
wnermual Jeath soints of Dect. pullorun,

otreins W115 end C.

 

Growth in plain bouillon.

 

Terip e Tinie e

' WLS G
40° C. 10 mnSe + +
42° 1T | ! : + "
agen n + +
46°" 1 4 +
4g°u 1 + +
50° 1 1 + +
62°" " + +
54° Tt 1" + be
56°" " + +
58°" " ~ +
60°" 11 — -
6e°" 1 _ -
64°" " _ _
66° t 1 _ + *
68° " " _
70° 1! 1! ~ —-

 

® . ge . ,
The charecteristics of the growth in this tube in-
dicated contaminetion, which was zsroved by u.icroscopic exam-=-

ination and the agglutination test.
wl Om

The slight cifference in the resistance of the two
strains cennot satisfactorily be explained. Azar Slants were
made from the last tubes of the two strains showing crowth
and tne agglutination test run to meke sure thet the "end point"
haa not been obscured by some contamnineting organism. Both
growtns proved to be pure cultures of Bect. pullorun. It is
possible that the findings would have been reversed had another
test been run, or thet the inherent resistence of the C0 strain
to heat is slightly greater than that of the W15 strain.

itesistance of Bact. pullorum to Some

, Common Disinfectants.

In view of the fact thet the question es to whet con-
stitutes the best disinfectant for use in incubators and scoops
harboring Bect. pullorum frequently is asked, and also because
no comparative tests of verious disinfectants appear to have
been made it was considerec well worth wnile to run tne follow-
ing experiment:

1/10 c.c. of & vigorous broth culture of Bact. pullorum
24 hrs. old wes nut into 5 o.c. of disinfectant and mixed well.
At the ena of 320 seconds a Loopful wes removed and rut into
5 ceGe Of plein broth. At the end of one minute another loopful
was removed and placed in a secona tube oi broth. This wes ree
peated at 1/2 minute intervals for six minutes. The tubes of
proth were then incubetcd at 57 C. for 48 hours end examined
for growth.

The contents of cll tubes were evenly mixed by gentle

shaking and the usual precautions were teken to avoid contamina-

oe

tion. See table for results.
-ll-

 

time in minutes.

 

 

 

 

Disinfectant

1/2i1i1 1/ej2\2 1/2)3 3 1/2/44 1/2 5 6 1/28 6

|

|
1:1000 HgClo. - |- -|- -\|- -/|- -|- ~|-

. | -
5’o Phenol. - |\- -j|- -|- ~ | - -~|- ~|-
1% Fhenol. + \¢ + \+ +) + +] + +|- |.
2% Idquor cresclis{- |- -j|- -|- -|- -| -| —
compositas. .

 

 

 

 

 

 

 

 

 

 

 

The experiment indicates that Bact. pullorum yields
readily enough to some common disinfectants when used in the
dilutions generally recommended, and the disinfection of coops
and incubators, as far as Bact. pullorum is concerned, should

not prove a serious undertaking.

The Longevity of Bact. pullorum
on Litter.

Three flasks of 250 cece capacity were filled about
one quarter full with a mixture of equel parts of clover hay and
wheat straw. The material was cut into lengths of approximetely
1 cm. to facilitete renoval of samples. The flasks were heated
in flowing steam for one hour on four consecutive days, at the
end oi wnich time samples were carefully removed and added to
tubes of plain broth. ib growth resul thagat the cnd of 72 hours
incubation at 37°C. the material wes considered sterile.

Two 48 hr. agar slant cultures of Bact. pullorum were
washed off with sterile physiologicel salt solution, the suspen-

Sion -- about 8 c.ce in amount -- divided ecually vLetween two

flasks and thoroughly mixed with the litter. The third flask,
ree ee

slo=

or control, received 4 c.c. of sterile physiological salt solution.
The flesks were kept in the laboratory end received diffused light
durin; the day, but no direct sunli,nt. pemples were removed
once a week end placed in bouillon, the resultin: srowths being
exemined in nansing drop and furnishin;g material for inoculation
of eé.er slants. Growth on ener wes cérefully exemined, and from
time to time suspensions in pnysiologicel salt solution were mace
end a;glutination tests run with positive sera,

At the tine of teking sarples, ebout 2 o.c. of sterile
distilled water were acded to eech flask.

See teble for results.
~lZ=

Broth culture, end Azar Streax Therefrom.

 

 

 

 

 

Date Flesk l. lesk 2. tlask 3.
: . (Control )
Oct. 14, 1916 Characteristic of Characteristic of Ib growth
Bacte pullorun Bact. pullorum

1" Pal " | WY | " vw

" 98 1" 1 1 "
love 4 " " " "

Tr? | 1 1 Ww 1 Wt rT

nm 49 n 1" i 1

ny rt " 1 r"
Dece 2 " " " Bo eabtai:

9 1 rT 1" No q nowt!

nm" 46 rt " " tt

m 9g 1 1 " i
Jéne 4, '17 " " "

at 11 tf ¥? tt iT

" 18 1 " tt N

m 8695 1" t n n
Febe 1 " " " "

tT | 8 rt Tt Ua rr

" 15 #" lb growth " "

" 8699 1 i" 1" "
Were Ll" " lio growth "

" go" " Contaminated "
Apr. 10 " " lo crowth "

rt : Ll W WW " W

i n i"

12

 

Contaninated

 

Contemineted

 
Agglutination Test.

 

 

 

Blask l. rlask 2.

560 7 13100 1:200 1:50 1:100 1:200

t + + + + +
lot run.
Ww Tf

+t + + + + +
lot yun.

t + + + + +
Iiot ynune
vy TT |

} + + + + +
Tot xyun.

+ + + + +
lot yun.

> + + f- + +
lot wone

b + + + + +
Not nun.

b + + + + +
lot yun.

+ + >

 

 

 

 

 

 

 

 
-14-

In the lest inoculation, sterile troth was poured
into the flesks in suificient quantity to cover the litter.

aS the detes show, the e:yperinent wes interrupted
for fully a u.onth et the end, but tre results probebly were
not effected, since Bect. ~yullorum hed not been isolated in
the weeks inmediavely preceding. Period of uniform isola-~
tion = 16 weeks.

The results indicete that Bact. pullorum would per-
sist for a eccnsiderable tiie on conterinated litter and em-
phesize the importence of tnorough cleaning ana disinfecting
of coops and incubetors thet heve Levborea chickens suffering
from White dlarrkeé. Low the lonvevity or the orgenismn would
be eifected by less ertificiel conaitions -- e.g. by the
presence of ler.e numbers of other vicroorgenisris -- is problen-
atical. Hperimentse to acetcrmine this point would be valuable,
but the ovsecuring of the celicete crowtn of Eact,. pullorum by
Other orgenisms on a,er would weke its cetcection difficult.

The Av jlutination Test es a Leans of vetection

of Carriers of Bect. pullorun.

securing blood sample: Tne followin, rethnod hes been
found convenient for drewing a sarple of chicken bloode-- Tie
the less cf the bird together end ley the fowl on a box or table
of convenient hei,-ht, on the right side, tne head being next the

Operator. Lift the left wing end strip the feetiers from the

—~<

under cidee Wioisten the skin Vv

a

ith 5) phenol end locete the

vené ulneris neer tne joint of the wins tip. With @ very snerp
=~] 5

knife, at one cut if zossible, slit the vena ulneris longitudinal-
ly tor ebout 1/8 inche This will ordinarily c,ive a suitable
enount of blood -= vize sbout 4 Gec. The wound is then moistened
with phenol and the bira given its freedon. If the flow is voor,
ley the bird on the other side and tap the right wing vein.
Occasional excessive bleeding shovlad be checked with a pledget

of absorbent cotton. The blood should be collected in a sterile
centrifuge tube clearly marked with the number of the bird, the
Clot broken with a sterile platinum needle and the tube set
overnight in the ice-box to allow the serum to separate out.
Centrifuging is not ebsolutely necessary unless it is desired to
test immediately.

Serples drawn in this manner cause the bird no perma-
nent harm, whatsoever. The wound heals very rapidly and the
feathers are cuickly restored.

Preparation of test fluid: acer slants were heavily
inoculeted with strains of Bect. pullorum suiteble for the test.
The slants were incubated for 24 - 48 hours at 37°- 38 C. and
the growths weshed off in sterile .85% salt solution. The fluid
was then filtered through sterile cotton to remove clumps, and
sterile 85%0 salt solution was added until a distinct but not
excessive turbidity resulted. (A turbidity corresponding to
that in tube 2 of icFerlane's (15) nephelometer is suiteble.)
Enough phenol wes then added to meke 5%, and the fluid well
shaken. It wes then stored in brown bottles in the ice-box
and would remein usable for a considereble length or tine,

The Test: The serum wes diluted with the test fluid

to 1:50, 1:100 and 1:200. A dilution of 1:20 wes at first used
~16-

es a base, but this wes leter abendoned end a suitable amount of
serum added with a capillary pipette directly to the test fluid,
Three tubes of about 5 ¢.c. capacity, containin: 2 ec,.c, of the
test fluid each, and correctly numbered, were placed horizontelly
in a step rack. To the first tube was acded 4 coc. of Serun,
to the second.2 cece, and to the third,]l c.c. The tubes were
then shaken end incubated at 3?°for 24 hours. & positive test
is markcca by the organisus flaking together end settling down
tovercs the bottom so thet the supernatent licuid looks more or
less clear. The resection is often observeble in 4 hours. It
is well to run e control of 2 c.c. of test fluid sinus serum
End another of tubes to which known positive cerum hes been

~

aede

0%

Comparison of Strains for Agslutinability.

This experiment wes conducted with a view to determine
the best strein, or conbination of strains, for the agglutination
teste Toe test tluics were prepare. es already outlined, the
polyvelent fluid bein, a rixture of ecual portions of the others,

assembled before sheking.
al =

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table le
Dilution.
Test rluid. perumMe ; : |
: } : | | :
o! 3' 8. 8) 8: 8; 8: gi Bg
Ql aj; Bi Bl wy, Bf] ©) BH] ©
rt ay re ri r r r- rr r-
|
Al Positive W.A.C.55}] +»! +) +¢/)4i)+4) +1 €: 4 +
AZ " +) +e +!) 4] 4] +4] Bi -| -
WLS " +1 e+; +] e] t+] t+] +) *] +
15 " +) ei rei ei +e] +! Pl =] -=
C n +i e+ | +] +i +] ¢] +] 4] +
Polyvalent " + +- + + + + » + +
Teble 2.
| — —
| Dilution.
| TT 7
Test Fluid. oerume . }
: 2} 3/8 3;/ 8| 8) 8 8
a) ad}a]a} a} a} ai a
dt) fd|aleal!] ala 4! 4
| __
Al Positive 55 +> *» as + + P ~ | «
AS " -_ oo: = on - _ -_ ane
RL5 " = | = -_ - | - -_ | om | ms
C " up > op P os = ome _
Polyvalent " + | + | + | * + | + + | P

 

 

 

 

 

 

 

 

 

 

P = Partial agglutinatione

,
 

-———

teen
~18—

In tebles ¢ end 4 the same anti,en wes used cs in tebles

lend 2, the positive serum bein: from cenother bird,

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Teble Se
Dilution.
Test luid. : verule : q
yt | | oO oO Oo; Oo Qo; Oo oO
, Of oO Oo: O'S] O11 G6 Oo
: PMP od Ss Ee RY St RL Sg
| oat ed 4 oti qalaql al wa
a L LL }
Tt rh |
Al Positive 55 !+ | + | + + + + + + +
AZ : " +i ple] el] bie | - | wd =
i
W15 i " +i ere] e+] &] ei e+] e ] ee] oY
x15 " +) +] +e] el] el e+] e | ] &
C " ee ee ee ee ee
Polyvalent " 4+: 4/4] | +] +] te] +]
. \
table 4.
_
| Dilution.
,
: {
Test Fluid. : perume OoO!lo!io9 oi oao!loalo °
. 4 oO, 9] OO} oO Oo' oO Oo Oo oO
i Ol Of}; A] wi ~st pl] oO] of fe
3 Qld) ad) dy dy dy) ay oadiod
| dj Al Al Atl Al Atl Al Ala
[ __
Al ! Positive 35 + + Pie} - - - - ~
AS ! " -_ -— = —_ —_ -_ om a= =—
W15 " + ie lel oe tw] ot -] =] -
|
|
|
C " + [+i ei eile le) &] Pl
| |
Polyvalent " + + | +i+i{i+4t!4¢4i{+i/] P| P

 

 

 

 

 

 

 

 

 

 
 

 
=19—

The test indicated that any one of the strains was suit-
able for diegnostic work, since all geve setisfactory eaegglutine-
tions beyond the 1:200 dilution. It is interesting to note thet
the polyvalent fluid was acclutinated in dilutions higher than
those in which the individual strains of which it was composed
were agglutinated. The seeming greater potency of one serum
over another for 8 perticular test fluid is also noteworthy.

This is well brousht out by a comparison of the ecelutinations
of test fluids W15 and Al. wl5, with serum 55 in 1:1300 dilu-
tion, geve a positive agclutination: with serum 55, a positive
result in a serum dilution of 1:900 only. Al, with serum 35
in 1:1000 dilution, gave a positive result; with serum 55
acglutination took place in as high a dilution as 1:1200,.

In the routine work of testing the College flock, &
polyvalent test fluid was invariably employed.

specificity of the Reaction.

Since Bact. pullorum hes been placed in the colon-
typhi group, it was consi dered worth while to determine whether
test fluids of the colon or typhoid bacilli would exhibit agzglu-
tination with known positive serum -- i.e. serum containing
antibodies for Bect. pullorum. The dilutions used and results

attained are shown in the tables.
 
Table I.

(Serum = positive 55)

gene

Test Sluid.

 

 

 

 

 

 

 

 

 

Dilution. _ :
Bact. pullorum. | Be typhosus. B. coli.
1:50 + - ~
1 +100 + - -
12200 + 7
13500 + - -
1:4C0 + - —
1:500 + - -
Table 2e

(Serun = nositive 35)

| Test Fluid,
Dilution. . : . —

| Bact. pullorum. i Be typhosus. B. coli,

| . _ ’ j (
1:50 3 ‘ - : :
13100 + ! ~ ~
13200 + - -
1:30 + - -
13400 + -
1:500 + - ~

 

 

 

In adcition, a test wes run in the sexe ailutions with a
serum containing no antibodies for Bact. pullorum by the a -slutine
tion test. In ell dilutions, the reedings were negative for both

B. typhosus end B. coli.
=2] =

The tests incicate thet there is no "group" agelutina-
tion and that the apciutination test, as used for the detection

mote
wo

of carriers of Bact. pullorum, is specific.

The Application of the Agglutination Test,

Some S500 niature birds of the College flock and of
verious breeds were tested. It was cesired to find out as fer
es possible how closely postenorten exeiinations of tested
birds checked with the findings of the agslutinetion test, to
cet some idea of the vercentage of reacting birds on a well-
kept plent in this particular section, end to cetermine the
convenience of the test when run on @ somevhat lerge scale with
all leboratory facilities.

From 6 to 18 perce nt of the birds tested gave positive
reactions. The percentage of reactors was lowest among the
heavier breeds, such ss wyandotts and Orphingtons, and highest
emnong the Leghorns. Whether these results indicate tnat the
Leshorn breed, by reason of its ectively functioning ovaries,
is more prone to carry Bect. pullorum than breeds tnat ere less
persistent layers, or whether the disparity is explainable on
other grounds, is not known. It is worth stating trat of 55
birds of the Leghorn breed comprisine & well-kept private flock,
” birds only, or ebout 12%, reccted to the test. some male
birds gave partial reections, thus raisin; the question whether
male birds can become carriers end, in turn, infect feneles.

It is a netter for regret thet reectin; birds were not

segregated, wintered over anc their ecgs incubeted. some inter-

estin: end valuable deta could thus heve been secured. The
m2 Due

Collese flock, while not kept for commercial curposes, neverthe-
less could not be utilized even in creater pert for experimenta-
tion on becillery white diarrhea alone The majority of the
retcting birds were sold Sor the teble end it was impossible to
cet complete cata on the percentage of infected ovaries,

One hatch of some 200 eg.:s from infected nens yielded
only 20% of chicks thet grew to maturity, many chicks dying in
the shell towards the cloce oy the incubetion period, This ex-
periment wes neither run nor supervised by the writer.

This year, 1917, the mortelity emons young chicks from
bacillery white diarrnea has never assumed serious proportions,
The menecement of the young chicxs nas been on @ par with thet
of previous yeers xnd in all probebility the weedin: out of
cerriers in tne tell of 1916 hes meterially lessened the loss

ancong newly natcied birds from bacillary white diarrhea,

The Complenent Bixetion Test in Diegnosis

of Bect. pullorum infection,

The epplicability of the complement fixation test to
the detection of carriers of Bact. pullorum is considered not
only of scientific interest, but it was realized that the test,
if religble, miz,ht prove very useful in cleerin; up doubtful

reactions by the «; ,lutinetion test.

,

rreperation of the seasents:~

Antigen (lst method):  4gar slants were inoculated
éna the growths wasned off exactly es aescribed in the prepare-

tion of test fluid for the agglutinetion test, except that the
turbidity of the suspension was adjusted to thet of tube 4 in
liecFarlane's (15) nephelometer.

®

Hemolysin: & CeCe of weched sheep's blood corpuscles
were injected into the vescina ear-vein of a healthy rabbit
four times on alternate days A week after the last injection
the animal wes fastened back down on an animel boerd, the fur
over the heart clippec short, the skin moistened with 5% phenol,
the heart located, end a medium-sized needle that had been im-
mersed in .5% phenol end flushed with sterile physiological salt
solution just creviously, inserted between the ribs, through the
cerdiac notch and into the left ventricle. About 30 c.c. of
blood were drawn off, the needle withdrawn, the visible puncture
phenolized and the animal given its freecaom. The blood clot
wes then broken up end allowed to stend for 24 hrs, in the ice-
boxXe at the end of this time the serum was poured off, heated
to 56° Ce for 1/2 hr. to destroy complement, enovgh phenol added
to neke 45%, end the hemolysin stored on ice until needed, In
the test, 1/2 Cote or the normal hemolysin wes eacced to 49 1/2
CeCe Of physiological salt solution, thus giving a Lio dilution,
- Blood cells (sheep). These were secured by tapping
the juguler vein of a neelthy sheep with a medium-sized needle,
after clippin; the wool close, end moistenin; the skin vith
phenol. About 60 Code of blood were drewn et a time, The
blood wes collectec in a stout flesk contelining sterile beads
and shaken until defibrinated, It was then poured into centri-
fuge tubes end centriiuged snertly vor 15 tins. Ghne serum wes

then drewn off, physiological selt solution added to about the

height occupied by the serum end the tubes, efter sheking arvsain

,r
Qe
 

 

centrifused for 15 mns. The salt solution was then pipetted

off and a fresh quantity added. This process wes repeated three
times, the salt solution being pipetted off the last time end the
cells stored in the ice-box in a securely stoppered bottle. The
addition of a preservative was found to be unnecessary if the
cells were all to be usec within 10 deys. In the test, & c.c,.
of blood cells were added to 98 c.c. of physiological salt solu-
tion, thus civing a 2)o dilution, which mede the test somewhat
more easily reed than wnen a 1% dilution wes used, end introduced
no undesirable factors.

Complement, | The guinea pig wes used as the source of
complenent and pielded so uniform a product thet no experiments
were tried with other animals. Tne method of drawing blood was
identical with that described for the rabbit, except that e
snaller needle was used. The blood =-- about 10 c.c. in quentity —
was collected in a centri fuse tube, the clot loosened with a
sterile platinum needle and the tube centrifveed for about 10
mnSe The serum was then pipetted off, diluted to 20% with
physiolocical salt solution end used at once. Complement kept
over night wes found to have lost much of its potency.

Positive serum. The method of drawing blood and
preparing cerum for tne compleme nt fixation test was the seme as
for the azsglutination test alreacy described,

suspect end nesyative serum, wren used, were prerered

in the seme manner es positive serun,
 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tubes.
xeacgentse . ‘
1 2 | 8 4 5 6 i 7
a Gees} CeCe CeCe Coe. 1G. Ces C0,
.857 IaCl solution 1e5°) 165 (1.5 1.5 11.5 ,1.65 11,5
20:3 Complement 02} .c4} 206 4.08 ' 1.0 .0.0 |} 1.0
1/0 Hemolysin o1C} .10] .10; .10 10 | 610 | 0.0
2,5 Blood cells .50| .50] .50|.50 | .50! .50!} .50
iieadings arter 1/2 hr e - + + > + - _
in 37°water bath.
+ * hemolysis. - = no hemolysis.

The titre of the complement was therefore .04, and this
reagent wes diluted so that .1 ¢.c. contained 11/2 times the titre -
1+@e 4 C606 of physiologicel salt solution were added to every

6 Cele of corplenent.

Nemolysin Titration.

 

 

 

 

 

! Tubes.
Reagents. | _
1 2 3 4 5 6 7 | 8
> Gece CeGe CeCe CeCe CeCe C.C, C.Ce CeC
6570 IaCl solution mee ft 165 11,65 1165°1165° 11.6.5 11.5 -} 1.5
' ’ ’ } , ’ .
20j0 Conplement -1C} .10} 10} .10/ .10] .10! .10! CC
1)0 Hemolysin Cl 02 C4 C6 208 |/1.0 | 0.0 |} 1.0
23 Blood cells ' 50! 250] 250} «50! .50! 250! ws0l 65
React ney Ss exter 1/2 hr. = % + + + + - -
in a7° water pet! lle

 

 

 

 

 

 

 

 

 

+ = henolysis. - = no hemolysis.
The titre cf the hemolysin wes

reea-ent we

S diluted so trat

theverore .C2, end this

el GeCe COntcinec 3 tines the titre.

,

Thus, 4 @.ce of physiological salt solution were eaced to every

6 c.c. of ner:01} Sin.

 

 

 

antigen Titretion. tb. I.
Tubes.

xeagents. —

1 2 3 4 5 6 7 8 9 10 Li

CeCeo| CeCe GeGe CoGe[CeoCe! GeCej/CeCef/ See! Sele |CeG,}] CeCe
Yalt solu- oo Hots : an pf
tion. 1.5 1.5 ile [ 165 {165 {1-5 {1.5 {1.5 :1.65 {1,5 {1.5
Positive
serume 002! 2.02: 2-02) 02) .02| .02|) .02)/0.0 {0.0 10.0 002
antigen. 002) 05} .08] 10] 15) 220} 225} 15} 220; .25/0.0
adjusted
conplenent. O.l | Q.1 |O-L JO.L [0.1 |O.1 |O.2 [0.1 {0.1 10.1 | 0.1

 

 

 

 

 

 

 

 

 

 

 

 

 

Incubatec 11/2 brs. in 37°C, water bath, then Hemolytic
eySterm added -=

 

 

 

 

 

 

Tubes.

geaze nts. :

1 & 3 4 5 6 7 8 9 10 Lil

Cole] Cole {Gee Gee [eGo [CeCe | Cole (Sele iGeGe |GoG,'|0.Ce
Adjusted pope fe fe pe Pfs oo
hemolysin. e1lO 010 «lO el O eLO eLO eLO elO eLO eLO eLO
2% 0 Blood !
cells. 250 050; «50; 50] 250; 2.50} .50 250 050} 50 edC
neadings &
after 1/2. + + + + + = - - - - }
hr. in 37° i
water bath. :

 

 

 

 

 

 

 

 

 

 

 

 

= henolysis.

no hemolysis.
27

These results were Guplicated in a titration run immed-

iately efterwards and indiccted thet the antigen possessed anti-

complementary properties, since control tubes 8, 9, and 10 failed

to show hemolysis.

Antigen so tnet the controls nisnt show henxolysis.

It was, therefore, decided to dilute the

Accordingly,

the 4antigen was diluted to correspond with tube 3 of MacFarlane's

nephelometer, end titratec, using the same amounts.

Antigen Titretion. IWOe Be

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tubes e

reagents.

1 L 3 4 5 6 7 8 9 10 LL

CoGo| Geel CoCo] GeCe | Gee |Gele|GeGe| GeQel CeCe CeOe | Bee
Salt solu- | | > | oof foe Pe ef fe TP
tione 1651125 11-5 {1.5 |1.-5 11-5 11.5 {1.5 {1.5 1,5 1,5
Positive
serum. eO2| 202) 02) .02| 2.02) -02} .02);0.0 10.0 10.0 e Ok
Antigen. .02] .05] .08/ 210] .15| .20] 25] 225] .20| .25/0.0
Adjusted
complement. |O.1 | O.1 |0O.1 |}0.1 |0O.1 |0.1 |0.1 | 0.1 |0.1 {0,1 /0.1

 

Incubated 1 1/2 hrs. in 37°C. water bath, then Hemolytic

system added --
 

 

 

 

 

TUDES «

Reagents. '

1 2 3 4 5 6 | 8 | 9 10 | ll

BeGol CeCe |GeCo [Cole [CeCe | Cole| CoCe/G.0-1/6.0,|0.C./C.C.
Adjusted : so
hemolysine el O eLO eLO «LO eld elO eLO eLO elO «LO eLO
2% Blood
cells. 050} 250; 250} 250] .50} 450; 250; -50} «50; .50} .50
xeadings
after 1/2
hrs.’ incu-| + + + + + + - + - - +
bation in
37°C. water
bathe

 

 

 

 

 

 

 

 

 

 

 

 

As will be seen from the table, this diluted Antigen gave

as unsatisfactory results as before.

It wes decided to deterzine

the anticomplementary dose or this Antigen and not to use an amount

Asa

greater than one-half of this

figure in titration.

Accordingly,

antigen and complerient were assembled as snown in the table, incu-

beted for the usual veriod end the hemolytic system added and incu-

bated as formerly -=-

Antigen Titretion. Wo. 3.

 

 

 

 

 

TubeSe

nease nts 4 _ .

L 2 3 4 5 6 7 8 9g LO Ll 12

Geel CeCe] CoCo | Cele [CoCo] CeCe] Cele | GeGe| CoC.) C.Ce [C.Ceol Cet,
Salt som] | mo
lutione {1.65 !1.5 41.5 {1.5 (1-5 |1.5 {1.25 {1.5 {1.5 {1.5 {1.5 {1,5
Adjusted !
comple= | .02 .02; .02} .02) O02} .0Z} .Oc}] .O&| 2.02) 202} .0E} 202
ment. ro : :
Antigen. aq ell ele eld e1l4 eld el6 el” 218 el9 ec eol

 

 

 

 

 

 

 

 

 

 

 
29

Incubated 1 1/2 hrs. in 37°C. water beth and Hemolytic

yvystem added, es in fables 1 end @.

 

 

readings
after
1/2 hrs. | + + + + + + + - - - -
incuba- -
tion.

 

 

 

 

 

 

 

 

 

 

 

 

 

The anticomplementary dose wes thus determined to be
ol6 GeCo Since one-half of this amount had previously failed to
prevent hemolysis in actual titration -- see Tables 1 and 2 --
there wes no object in re-titrating, since hemolysis obviously
would iave teaxcen place in all tubes.

This antigen clearly was worthless ror complement fixa-
tion work. It wes, however, prepared from a single strain of
Bact. pullorum, and the possibility of securin, better results by
using a polyvalent entigen wes recognized. (The necessity for
polyvalent antigens in dealing with typhoid fever, glanders, etc.,
is eryhasized by Kolner -16-). The monovelent preparetion was
therefore discarded and an antizen composed of five cifferent
strains of Bact. pullorum prepéred. This entigen wes adjusted

to correspond to tube 4 in iiecFarlane's nephelometer.
antigen Titration. Ib. 4

=50—

A

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tubes.

reegents. ,

1 2 3 4, 5 6 7 8 9 10} Il

BeBe] Coe! Cole] C.C.1;0.0./ 0.0.1/0.0.,0.0./6.C~. 16.0. |C.G.,
Salt solu- | : :
tion. 1.5 '1.5 {1.5 |1.5 {1.5 }/1.5 {1.5 {1.5 {1.5 31.5 {1.5
Fositive
serun, 02, 02) OZ} .O2!) .O2) .02) .02/0.C [0.0 |O0.C °O2
Antigen. 02! .05} .08 .10; .15) .20, .25) .15; .20) .25)/C.0
Adjusteca
complement.|O.1 |O.1 ;O.1 |} 0.1 {0.1 | O.1 |O.1 {0.1 {0.1 ]0.1 |0.1

Heuolytic System edded in usual exounts efter 1 1/2

hrs. incubetion at 37°C. in water bath.
xeadings 1/2
hr. aiter
addition of | + + + + + + + + + + +
Lemolytic

system.

 

 

 

 

 

 

 

 

 

 

 

 

This titration wes run in duplicate.

A prelininery titretion, similer to that shown in Table

3, was run with the object of determining the anticomplementary
dose. This fector, however, did not e::ist with the amounts used,

t

nor did it sppear in the ectueal titretion. The rirst batch of

Hy

entigen wes prepered Trom growths grown on ager or very poor con-

Sistency and it is possible thet minute verticles of the recium
were vresent in the entigen to an extent sufficient to give this

reevent anticomplementary properties (17).

6
Though enticonplementery reaction was thus got rid of,
the antigen apparently was of no velue for the complerent Tixa-
tion test. Havins been tried with two other sera that were
strongly positive by the esglutinetion test with no tetter re-
sults, it was decided to e::periment with antigens of dirtferent
preparation.

,

Plasmolyzed antigen. A polyvalent entigen wes pree
peared as vefore, except thet distilled water was used instead of
physiolozical salt solution end the suspension reated for 1 hr,
et 60°C, It was then shaken by mechine for en hour more, after
the addition of sterile beads, This heating and shaking were
repeated on three consecutive devs, at the end of which time the
pacterial cells, as ~roved by mieroscopical examination, were
efficiently ruptured. It was hoped thet the bacterial anbocep-
tors would be anchored by some substance in the bacterial cell
that had diffused into the suspending fluid, or had become aveile
able by Simple lysis of the cells.

This antigen was given a thorouz;h trial, being run in
duplicate with one positive serum, once with another positive
serum, and once with a negative serum. The amounts used ran
from .02 cece to .25 c.c., as usual. |

| In all tubes, with all sera, henolysis took place,

Before the entigen was discarded, enough 20% salt so-
lution was added to give «85%. me presence of electrolytes
bein: necesse ; for acclutination, it wes tiougnt that, possibly,
the absence of salts in the entigen wes responsible for the fsil-
ure of the reagent. On titrating, however, no better results

were secured tnan before.

The uniform failure or all antigens conteinin: suspen-
sions of the organisms pointed to the necessity of trying other
types.

| filtrate from broth culture: sive bottles, eacn hold-
ins approximetely 50 c.c. of plain broth, were inoculated with
five different strains of Bacte pullorum end incubated eat 57°C.
for 72 hrs. The broth wes then filtered through a Berkefeld
filter by suction, the clear filtrate cerbolized to .5%, shaken
end put on ice.

This antigen wac then titrated, the usual amounts of
reazents being employed. The entigen thus rangea in emount fron
02 @.c0. to .25 2.C.e Hemnolysis occurred in all tubes except
tnet containing 25 CeCe The set wes put on ice over night am
the bleod cells settled ont, there beings no indication of hemolysis.

A titration with the seme serum was now run, using large
emounts oxy the antigen, other reagents beinz added as usual, as
shown in the teble.

Antigen Titration. Ib. 5,

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tubes,

Reagents. _ :

L 2 3 4 5 6 7 8 9 10 | ll

C.C. G.G. C.C. CG.C. G.C. G.C. C.Ce C.CG. C.C. G.0, CG.C.
salt solu- | vo
tion. 1.5 ;1.5 {1.5 {1.5 [1.5 {1.5 {1.5 {1.5 {1.5 {1.5 |1.5
Positive
serum. O02; .02| .O2; .O2| .02/] .02} .02); .0C;} .00] .00| .O2
Antigen. ol 015} .20! «25; .30| .35) .40/] .30} .35) .40! .00
Adjusted
complement. /O.1 | 0.1 | 0.1 |0.2 |0.1 {0.2 | 0.1 |0.1 |0.1 |0.1 | 0.2

 

Incubated in 37°C. water bath forl 1/2 hrs. and Hemo-

,

lytic System added as usual.
 

 

L
MH a
xeadings
efter 1/2
hrs. incu- | + + + P - - - + + + +

betion with
Eenolytie
System.

g

 

 

 

 

 

 

 

 

 

 

 

 

The titration wes run a second time, results being
duplicated.

So far, this antigen had yielded somewhat encouraging
results. It was, however, realized that the antigen would heave

to meet several conditions before it could be pronounced successful

le. Yield negative results in an extended series of
' titrations with sera from normel birds,

ge Yield positive results in a Ssiniler series with sera
' from infected birds.
(Normality or infection of birds to be deterimineé
by autopsy as well as by acelutineation test).

4. Be capable of concentration so that handier amounts
could be used. (Hot absolutely necessary).

48s a preliminary step, a titration was run using plein
proth in plece of eantigren. The emounts employed were the same
as in Teble 5, tnus ranging from .10 c.c. to .40 c.a. Other
reagents, incubation period, and incubetion tenperature were as
usual.

| Complete hemolysis occurred in all tubes in 1/2 hr.,

provin; thet the fixation or the complement in tne previous tests
was not due to any substence originally present in the bouillon.

At this stage, complement fixation work was interrupted

for the greater part of a week. perum wes tien prepared from ea

blood semple from a vigorous fowl which hed not «cacted to the
ecerlutination test. AS & precautionary measure, the test of
this serum was run with tnat from the fowl whose serum ned reacted
positively in the previous tests. The amounts of reasents used
were identical with those pefore employed, as Shown in Table 5.
Both titrations showed hemolysis in all tubes,
tienolysis in the ‘tubes which had received positive serum
wes altosether unlooked for and it wes thought thet some error of
technique hed crept into the test. There was, also, a bere pos-
sibility of the serum no longer possessing antibodies (18). he
titration was, therefore, re-run end en esclutination tes cet Upe
47.

ve titretion chowec the same recults es before ana tre apclutine-

tion test shoved distinct o glutination in all t::sce dilutions eat

a

e
5 1/2 hours. An additionel titration run With a
wuifrerent positive serum ,ave negative reauin-s in all tubes,

There seemed to be but one interprctction to put on

metters —— namely, that the antigen hea lost tne power of fixing
enboceptor and complenent which it hed formerly, to a rather feeble

extent, possessed. anotner cuentity was therefore prepared in a

Similer manner end at once tested out.
This antigen proved to be even feebler than the first,

though the reactions when lerge cuantities were used with positive
serum were specific enougne A test with two negat

,

necetive reeacings, but tre worth of the antigen disapreared at the

i

ive sera eve

end of three days, despite the fect thet it wes kept on ice in a
securely stoprered brown bottle.
Hollowing these experiments, various tatcnes of antigens

were preperec and tried with erratic results. some were worthless,

ty

Others acted in very lerge quantities only, while some gave clear-

*

C

cut reections in smaller amounts. sepeatec tecting bore owt the
fact thet no entizsen would keep longer than four days. wnetner
such en entigen would lend itself to concentretion or precipita-
tion of the active principle (19) which would keep for some
length of tine could not be determined.

The results of some other oxperinents on complement
fixetion work in the detection of Bact. pullorum infection in
fowls came to hand efter the e-cperiments before cetailed had been
completed. A limited mumber of tests, the entigen used beinz,
presunebly, a nonth-old broth culture of Bact. pullorum, wes con-
ducted at the U.S. Dept. of Agriculture (20) with uncertain re-

’ ’ ’

sults. The findings of Gege (21) are no more satisfactory.

The Intradermal Test.

The possibility of developing a test similar to the
tuberculin test in cattle for the detection of cerriers of Bact.
pullorum easily suggested itself. Prom time to time, sbout 1/4
G.c. of bovillon cultures of Bact. pullorum of 24 hours! to
l week's incubation, killed by heetin: to 60°C. ror ] hour, were
injected into the wattles end combs of birds positive to the age
slutinetion test. liecetive re:ctings birds were Sinilerly in-

jected as controls. lo satisfactory reactions bein; secured
from these tects, it wes cecided to lensthen the ceriod of incu-
bation to at least one month and prepare a test fluid by the same
method used for the production of tuberculin.

At this time a publication dealin: with tne cubject wes
received from tne Bureau of Animal Industry. The euthors had
Securec food results from a bouillon culture incubated for one

month, killed by heatin; to 60 C., phenolyzed, and xent on ice
for six months when it wes used without further preperation.
Filtration or concentration, according to their rindings, aia not
Seeri to enhance the value of the fluid for diggnostic purposes.

The bovillon culture being incubeted by the writer WES
accordingly kept for one month at 37°C., killed end phenolyzed
as before rentioned, end sect in the ice box. After 2 weexs!
storaze, cbout 1/4 c.e. were injected into the wattles and comb
of a strongly positive chicken with negative results. The in-
jections have been repeated at intervals of about two weeks to
the date of writing. The test fluid is now two months old.

three more cultures, all polyvalent like the first, but
differing slightly es to strains involved, have also been en-=
ployed without success.

The nesative results secured may be due to lack of
"eceing™ of the test fluid. Just weet effect this could have
is not clear. There is also the possibility of the strains em-
ployed heing unsatisfactory.

The intradernal test for the detection of Bact. pullorun
carriers may be regarded as a new and interesting study for the
pathogenic becteriologist. The real worth of the test can only
be gavged after a considereble period of time when any difficul-
ties incidental to the preparetion of the test fluid as now mede
may be justly avpreciated, or obviated by the adoption of other
methods, and a correct estimate of the convenience and reliability

or the test in vield work errived eat.

‘
The Infection of Mature rowls.

An important consideration in the Bect. pullorum carrier
problem is whether healthy, nature fowls can becone infected and,
if so, in what manner infection occurs. The researches of
Rettger (22) indicate thet infection follows prolonged contact
with diseased birds, as well as contect with contaminated litter,
and the eating of contamineted food. The question seemed to the
writer important enouvsh to varrent experimentation. Chenges in
the College flock made since testing rendered the supply of
reactors very low anc it was decided to postpone a "ccntact" ex-
periment for the tine bein. twelve fowls of mixed breeds were,
however, put in @ roony pen “or a feed end litter infection ex-
perinent. These were tcsted immediately by the agplutination
test ena found negative. ten dezys later they were eseain tested,
with tne same recult. Besinnin: in the middle of January 1917,
ebout 50 cea. of a 48 hour bouillon culture or Bect. pullorum
were daily scattered over tne litter end into the feedins trough
and drinking watere The birds were tested weeily, the pen
cleaned out whenever it becare dirty enough tofrpossibly tnjure
the health o the birds, and the attendant exercised the precau-
tions, such as cnangin; poots, ete., usual in a test of this kind.

The results of the experiment ere embodied in the ap-

pendec table.

’
 

Final .cclutination Test on June 1, 1917.

 

 

 

4eclutination Test,
Bird Ilo. , Breed. _
1:50 13100 13200
D 22 Leghorn - _ ~
D 617 Brd. Rock - - _
C 855 moon . _
C 364 nom + P -
D 607 moon - . _
D 936 mom . _
D 641 R. I. Red - ~ -
D 658 oR - - 7
D 663 A + + +
D 651 " + +

 

 

 

 

 

Of the l2 birds, one died; another was usec as a control
in the intradermal test.

fowls D 663 end D 651 first gave a positive reaction in
the begimnin; of May. C 364 resected positively towerds the end
of lay.

| The experinent was interrupted durin; the month of Lliarch.

A notewortny feature is the epperently rapid rormation
of antibodies after infection. All three reectors were cleerly
negative the week before their serum géve a positive reection,
which in the case of two then showed in all three dilutions.

On post-mortem exeninetion, all three fowls showed

typicel ovarien infection with Bect. rullorun.
 

 

 

+ —
ving the first three montns of the experiment the
weather wes unusually cold, the temperature of the pen frecauently
approeching cero Han. while the health of the birds did not
suffer, owing to the pen being well ventilated and dry, quicker
results would, perhaps, heave been secured had it been possible
to delay the ezperiment until wermer weather,

the experinente corroboretes the findings of Rettger (2£5)--

nenely, thet grown fowls may become infected with Bact, pullorum
through the ecting of conterineted FO0d » Too much cs sre cannot,
therefore, be telzen to keep down the spread of bacillary white
diarrhea in this Wey The custom prevelent amon; some poultry-
men of feedin, egss thot have feiled to hatch to their fowls is

Specially to be conderine d. Even the small pieces of eggs Shells

ct

het cover the incubator trays aiter hatching should be disposed
of in ea fashion to preclude the possibility of their being eaten
by fowls. With dead ebryos, they Should either be burnt or
buried deep with quickline the prectice of darkenin; the in-
cubator as a hatch comes off, to prevent the young chicks from
pecking at shell frepments, etc., is undoubtedly sound.

Pathogenicity of Bact, pullorum

for Experinental Aninels,

This phase of the study of Bact. pullorum, oving to
rather serious losses amon: the leboratory animals and the ined-
visability of introducing fresh stock until the trouble hed been
cnecized, did not receive the emphasis originally intended,

Four guinea figs were fed about 30 a,c. of a 48 hour

,

brotn culture of Bact. pullorum four or five tines 4 Weel, The
—1 Om

culture wes mixed with their drinkins water ena food, The ex-=
perinent extended without interruption from Avril 19 to June 6,
the sera being tested from time to time for agglutinins. At no
period was a positive reection secured by tne o--lutineation test
in dilutions of 1:50, 1:100 and 1:200. On June 6, tne guinea
pigs were killed. All orgens of ell enimels appeared normal on
post-mortem exendnation,

The test was not fer-reachins enouch to werreant the
drewins of any positive conclusions. It is realized tnat a
lerger nunber of animals shouid have been red over a considere
ably longer period. liore recently isolated strains tnan those

employed nignt nave proved more desirable,

The researches of Gege (24), who found thet Bact, pul-
lorum is tozsie for ,;uine& pigs, rabbits, and rats, are exti rene ly
interestin;. This investicetor reises the cuestion es to whether
eogs infected with Bact. pullorum are dangerous for hunan con-
sunption. :

The Resistence of Bact. pullorum in

Infected Esrcs to Coo! rinse

Fresh eggs were immersed for three minutes in 1:10CO
HigClp and 50% alcohol. The blunt ends were then flemed and each
esg inoculated with about 1/10 o.e. of Bact, pullorum in physiolos-
ideal salt solution. Precautions were teken to keep syringe and
needles sterile. The punctures were filled with collodion and
the esos incubated for periods ranging from 24 to V2 hours. They

were then immersed for verying periods in boiling water at 99°C,,
being placed in a dish containinzs cold 5053 alcohol immediately
after removal. mne yolk wes then emptied into a lovy jar con-
taining ebout 40 G.c. of sterile bouillon and incubated for

48 hours. At the ena of this period agar streaks were made,
the -rowths thereon examined microscopically, end agglutination
tests run.

Conteninations were not infrequently encountered, but
the results of the experiments may be briefly siwamerized as
follows:

In no case was a culture of Bact. pullorum recovered,
directly or indirectly, from any egg that had been boiled for
more than 1 1/2 mns. Little difficulty was experienced in re-
covering the orgenism from eggs that had been boiled 1 minute.

The condition of the eggs after 1 1/2 mns. boiling is

whet would be described as "soft boiled",

Experiments with eggs differently cooked were not con

pleted,

| In view of the fact that esses naturally infected with
Bact, pullorum contain but few organisms, and since the organ-
isms appear to be killed in the process of cookins eggs to a
"soft boiled” condition, the writer is inclined to the view
thet, even assuming the toxicity of Bact. pullorum for ran, tne
naturally infected eggs undoubtedly on the market dco not con-
stitute an appreciable danger to public health.

,

Additional iemarks,

Some interesting and importent phases of the study of

Bact. pullorum intended to be touched upon hed, for lack of tine,
 

to be left alone. The writer refers perticulerly to the role of
the male bird in the spreed of Bect, pullorun inieoti on. Partial
acolutinetions in low dilutions have been observec. from ‘tr 70 mele
birds of the College flock. wnetner infection becomes localized
in tne reproductive orgens of the male bird which, in turn, can
infect females is ea auestion which, to the best of the writer's
knowledge, hag never been investigated end offers score for inter-
esting experimentation.
rhe resistance of Bact. pullorum in infected egss to

cooking was intended as a preliminery step in a feedings experiment

of

involving several of the starr of this Dept.

The problem of preperins en antigen suitable for use in
the Complement Fixation Test could perhaps be solved by meking
an extract of infected ovaries, or even of normel fowl tissu
such as the liver or heart. The feilure of all antigens employed
up to this time is rather strilcing end stimulates further research,

while infection with Bact, pullorum must inpeir the ecco
aying aualities of a fowl, an infected bird may, to ell appear-
eanees, appeer perfectly healthy. The writer had two recaetors under
almost daily observation Yor one veer and never could detéct any
‘ns in the behavior of the birds thet would indcieccte infection,
In the College flock, also, where sickly birds are et once serreras
ted, 12% geve positive reactions,

Tne writer estimates thet the total time necessery to
arew a sample oF blood from e« fowl under orcinery conditions is
under 5 rns. Chus, «at least lz birds cen be bled yer hour. che

~y-- 47

ectuel time to set up an Egslutinetion test, ever;

4

ciines being reccy,

is very smell, but the preparation of glcecewere, cerun, test-fluid,
etc., consvimes time more Gittieult to teccure. with vlood
Saroles sent into the leboratory, vrobebliy 1CC secules eorld be

run Gcily; if the operetor hed to collect his own Serples, the
nuiber would, of course, be ruch siialler,.

sum.cry cond Conclusions.

le Becillery white dierrhea hes obteinea a foothold
,
t.

nieny of the commercial poultry plents in et least the Southern
pert of Lichigen. ane e::tent to which the adisecse hes invaded
ordinery Tern flocks in uncertain, but is not, on the whole,
Serlouse
| 2e Beet. pullorum will probebly pers:ist for & conside-:-
able tine on litter, but yields reacily enough to the common dis-
infectants.

Oe iO Satisfactory Complernent Fination Test hes been
devised to cetect carriers or Bact. pullorun,

4. ‘ne Intracernel Zest bes not been used sufficiently
for its value to be determined,

5. Tne A2lutinetion Test is undoubtedly of velue in
the detection of Bact. pullorum cerriers end much good hes been
accomplished throvgn its employment. (See Report 6, 1917, of
storrs Earp Stn., to nand at this wreiting.)

6. Mature fowls ney become dangerous cerriers of Bect.
pullorun from eating food centeminetec with this orgenisn.

7. There is no evidence to show thet a@ carrier ceases
to becorie such xeert, possibly, in e::tremely rare ceses, Hold-

ing over infected birds in the hope thet they moy free themselves

from infection is, therefore, futile.
Ahn

Ge nat any breed of fowl is more susceptible than
enotner to pacillary white diarrhea has never been proved.

9. There is no rreperation tret will safeguard young
chicks from white diarrnea if they sre exposed to infection. If
a chick has Bact. pullorum in the unabsorbed yolk, not?:in;: it mé.37
be given can nossibly reach the orsenisms; if the chick hes eaten
food ccntamineted with Bact. pullorum, nothing thet could kill the
organisms in the elirentary canal will leeve the chick alive, The
weeding out of ell meture cerriers, the proper disposal of dead
embryos, and thorough disinfection of coops and incubators is the
only wey to combcet the disease,

10. There is evidence thet Bact. pullorum, fed in liberal

quantities over @ considerable period of tine, is actually toxic
for laboratory animels,

ll. Dne to:sicity of Bact. pullorum for man is uncertain.

In any case, the thorough cooking of eggs probably would prove an

q

efficient sefecuerde
v
Aciznowledgments.

liy cordial thanks are due Dr. Giltner, H. Jd.
Sstafseth, and other members of this Departnent for advice
and sugsestions freely prorrered and inveriably found help-
ful liy appreciation is also extended to the verious men-
bers of the Poultry Departnent, past and present, for help-

ful cooperation in meny practicel details,
10.

lil.

rettger:

nettger:

Rettger

Jones:

retteer:

Gace :

ettger:

Jones:

Liorse:

Herzog:

Pernot:

ace:

Besson:

and Stoneburn:

neferences,.

Satal Septicemia in Younc Chicks.

Vol.
ited. Journal, 1900, p B00 « :

71, Het.

Further studies on Fatal septicemia in Young Chicks,
or White Diarrhea. Journal wed. Research, Vol. 2l,
1909, p 115. : .

Bacillery wnite Diarrhea of Young
Chicks. Bulletin lo. 60. Storrs 4g. Exp. Stn,
Annual Report of the IY. State Vet. Collese,
191 Qex11 , p 696 cof
Votes on Ovarian Infection with Bact. pullorun
(Rettger) in the Domestic Fowl. Journal lied.
search, Vole 24, June 1911, p 491 = 496.

Re=

Bacillary Thite Diarrhea of Youngs chicks. Storrs

Bulletin iD» 74. DeGe Lolz.

The Diegnosis of Infection with Bact. pullorum in
the Domestic Fowl. Mass. Exp. Stn. Bulletin 148,

1914.

Bacillary ithite Diarrhea of Young Chicks. Bulletin
77, 1914, p Bee Storrs.

The Value of the Macroscopic S¢glutination Test in
Detecting Towls that are Herboring Bact. pullorun.
Journal lied. Research. Vol. 27, To. 4, 1913,

p 481 ~ 495.

woiite Diarrhea of Chicks. U.S. Dept. of Ag. Bureau
of Animal Industry. Cirouler 128, 1908.
Disease-producing Licroorganisms, p 295 1L91LO
Lditione

An Investigation of the wortality of Incubator
Chicks. Oregon Ag. College. Dept. of Bact,
Bulletin 103, Vers 1908. ’

Chicks. Bulletin
auyust 1915, p 47.

DEC. pullorum Infection of Youn;
163, EES 6 Age Colles ‘Ee Lacp « Styne

rrecticel Beetex clolosy, ierobiolo-y, and Serun
Cherepy, p 54.

v
16.
17.

18.

19.

20.

Ble

Cee

Le
24.

LeBerlene's Yevheloneter =

Tube 1 = 99 perts 1js E2804 to 1 pert ‘> BaClo
" g2H298 =" "2 perte " "
tt 3 = 9” WT tt Ww W 3S rT Ww Y
EtG.

Lolmer: Infection, Immunity end Specific Therapy, 1915,
P 4896

t.00ker Antigenic Properties of Different Strains of B,
typhosuse Journel of Imm. Vol. 2, lk. 1, 1916.

Horton: apparent .iecovery of Hen from Becillary ‘ihhite

Diarrhea. Journal of Bacteriology, Vol. Ll,

io. 6, Lov. 1916.

Kolrner Infection, Tnnunity end Specific Therapy, 1915,
p 422 = 4235,

Ue Se Dept. of ag.: An Intradermal Test for Bact, pullorw:
‘Infection in Jowls. Bulletin 517. Teb. 16, 1917,
P if. : ! :

GEE 8 Eectreet fror. Personal Letter, Mar, 30, 1917, --
"I have found the Complene nt ' rination’ Test (for
detection of Bact. pullorum carriers) to be un-
reliable as earried ovt in my hands."

snettger: See ieference 6.
see ueference 6.

Gace; Toxicity of E:gs Infectec with Bact. pullorum.
Journel Exp. ledicine, 1916. 25, 475 = 489, :
— 4.8 -

PLATES SHOWING TYPICAL MISSHAPEN EGGS
IN CHICKEN OVARIES INFECTED
WITH EACT.PULLORUM.

Infected eggs sre usuelly greenish in
color, are relatively hard, end lack
the elasticity of normal ceywtoplasm.
— —

= ed =