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UNITED STATES DEPARTMENT OF AGRICULTURE

Ceniributien from the Bureau of Anima! Industry
A. D. MELVIN, Chief

Washington, D. C. PROFESSICNAL PAPER February 16, 1917

AN INTRADERMAL TEST FOR BACTERIUM PULLORUM
INFECTION IN FOWLS.

By ArcHisALp R. Warp and Bernarp A. GALLAGHER, Pathological Division.

CONTENTS.

Page. | Page

Dissemination of infection in white Comparison of resuits of agglutina-
drarehned 22 .e e S af | tion and intradermal tests_______ 12

Mie acchutination (teste 22. 2 i. 2 | Significance of swelling as an indi-
XpeLimental Work. =—2 oo Do CONICS GE IRR RON as 13
Tests of artificially infected birds 9). WMarioussbiologzic= tests2 22 === 14
Field trials of the intradermal Summary and conciusions_______~~ 14

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DISSEMINATION OF INFECTION IN WHITE DIARRHEA.

Of the numerous diseases to which poultry are susceptible it is safe
to say that bacillary white diarrhea is by far the most widespread
and most destructive. Its ravages are confined principally to baby
chicks, but it is the pullorum infection in the hen which is directly
responsible for outbreaks of white diarrhea in the chicks, since a
certain percentage of her eggs hatch infected chicks and the excre-
tions of these spread the disease to the other birds in the brood.
The exceedingly high mortality of white diarrhea, amounting in
some cases to almost 100 per cent of the hatch, practically prevents the
rearing of chicks in infected flocks. The disease is contracted dur-
ing the first four days of life, and deaths occur as a rule during the
first month. It has been demonstrated conclusively by several in-
vestigators that chicks which recover may carry the causative bac-
terium in the ovary and serve as a source of infection in the future.
Infected hens usually exhibit an ovary containing several angular,
hard, discolored ova; however, the organ may continue to func-
tionate and from time to time an ovum is released which harbors the
infective agent. Outbreaks of white diarrhea as a result of con-

Nore.—This bulletin is a report on a study of a disease of fowls that is quite de-
structive, and should be serviceable to those who are interested in poultry and poultry
diseases.

f2248°—Bull; 51(—17

a2, BULLETIN 517, U. S. DEPARTMENT OF AGRICULTURE.

sod

tuminated incubators or brooders could be controlled readily by sani-
tary measures, but infection through the egg must be prevented by
a process of weeding out the carriers among the hens used for
breeding. ;

THE AGGLUTINATION TEST.

Since the presence of the Bactertum pullorum in the ovary of the
hen is not betrayed by external symptoms, it was necessary to devise
a biologic method of diagnosis in order to detect the presence oi the
disease in the affected birds. The agglutination test was found to
be applicable for this purpose, and several agricultural experiment
stations have taken up the work on an extended scale, offering the
service to poultrymen at a price that barely covers the cost of the
work. This, in Connecticut, is understood to be 10 cents a fowl.
The work of drawing blood samples and sending to a laboratory is
necessarily tedious and relatively expensive as compared with the
value of a bird. A simpler, cheaper, and equally accurate diag-
nostic method would undoubtedly contribute to greater popularity
of this valuable work in disease prevention.

EXPERIMENTAL WORK.

The writers have undertaken to determine the possibility of pre-
paring a biological product from Bacterium pullorum to be used for
the diagnosis of the disease caused by that organism. The general
idea was to develop a diagnostic method somewhat analogous to the
intradermal tuberculin test, particularly as apphed to fowls.

TEST OF ARTIFICIALLY INFECTED BIRDS.

Two strains of Bacterium pullorum were planted in 1,500 c. c. of
plain bouillon in the amount of one loopful each. This culture was
incubated at 37° C. from September 19 to October 19, 1914. It was
then placed in the ice box until May 4, 1915. On this date 100 c. ¢.
of the culture was passed through a Berkefeld filter. The filtrate
was determined to be sterile by cultural tests. Carbolic acid was
then added in sufficient quantity to make a 0.5 per cent solution.

On May 17, 1915, two drops of this filtrate were injected into the
right wattle of a hen that had been injected with Bacterium pullorum
on September 22, 1914. The liquid was injected slightly above the
lower border of the wattle and no attempt was made to place it
within the layers of skin. Twenty-four hours later the wattle showed
an edematous swelling. ‘The following day, 48 hours after injection,
there was noted a pronounced edematous infiltration of the entire
wattle. A swelling of this size in other intradermal tests would be
considered as positive. The temperature was normal. On May 20
the swelling of the wattle decreased ccnsiderably, and 90 hours aiter

‘TEST FOR BACTERIUM PULLORUM INFECTION IN FOWLS. =

injection the wattle appeared normal. The wattle of a control, a
noninfected bird, injected at the same time, remained normal. On
autopsy the ovary of the infected bird presented several angular ova
typical of pullorum infection. A pure culture of B. pullorum was
isolated from the ovary. The result of this experiment suggested
that a diagnostic test might be developed.

Work with the same filtrate was continued by evaporating 100
e. c. to one-tenth of its volume in a water bath at the boiling point,
the purpose being to test the value of a culture filtrate containing the
products of the organism in a more concentrated form.

Twelve fowls were injected intravenously on May 27, 1915, with 1.5
ce. c. each of a 48-hour bouillon culture of six strains of B. pullorum.
About two days after inoculation the fowls showed marked symptoms
of illness—pale comb, drowsy attitude, and ruflled feathers. Five of
this lot died within a period of 26 days as a result of the injection.
On autopsy the following lesions were observed: Livers enlarged,
darker than normal, and covered with necrotic foci. Spleens were
enlarged and studded with necrotic foci. Ovaries contained irreg-
ular-shaped hard, dark-colored ova typical of pullorum infection.
Tn one case there was severe pericarditis and in another considerable
amber-colored fluid in the peritoneal cavity.

On June 9, thirteen days after the fowls were inoculated and while
they still were sick, blood was drawn for serum tests. Of these,
fowl 73 was injected in the right wattle with 0.1 c. c. of the con-
centrated filtrate. Three hours after injection considerable edema
of the wattle was observed.

Two control fowls, 74 and 75, supposedly healthy birds, each re-
ceived 0.1 c. c. of the concentrated filterate in the right wattle.
Three hours later they showed edema of the wattle, practically of
the same extent as that shown by the infected bird.

On the next day the swelling of the wattle of the control fowls
74 and 75 had entirely disappeared, while fowl 73 showed consider-
able swelling. This swelling continued to be marked at the forty-
eighth hour, after which it began to subside.

The results of this test are shown in Table I and include the re-
sults of autopsy, cultural, and agglutination tests.

TABLE I.—Concentrated filtrate tested on fowls 13 days after inoculation.

1 Controls.

A BULLETIN 517, U. S. DEPARTMENT OF AGRICULTURE.

In this second test, while the concentrated filtrate gave satisfactory
_ results, the reaction was not as marked as in the case of the first
fowl, on which the nonconcentrated fluid was used.

On June 22 a third series of trials with the filtrate was conducted
upon 6 fowls that had been inoculated intravenously on May 27
with a culture of Bacterium pullorum.

Fowls 77, 78, and 81, with two controls, 86 and 87, received in the
right wattle 0.1 c. c. of the same concentrated filtrate as used before.
In each case the injected wattles showed some edema within an hour.

On June 24 fowls 77 and 87 still showed slight edema, while fowl
Si showed a considerable swelling. The results of these tests are
shown in Table II, together with data on agglutination test, autopsy,
and cultures from ovaries made at a later date.

TasiLe Il.—Concentrated filtrate tested on fowls 26 days after inoculation.

| samara Edema | Edema

ee aiter1 | after 24 | after 48 gglu-
Fowl No.— Four! hours, | hours, Autopsy. | Culture. tiation,
| June 22. |} June 23. | June 24.
a Per ta nanege Pe Ese || a ee
CEL Be RE OS Ie 3 ae REO Pe ERO AEE | eee Slight. .| Slight. + + =e
USS. Sa ae oe A eS See de dente ese feos ose do. ie Fea) ae = + +
Sle cease sssgseeoSessseesecssecseeseserseed se- do.. = Slight.. le FE al ? + +
po GRAD Pie eee mwa eR A ernie el ane Ca ee a PaO | = ax ae
Oiler ss a ee Ss Sen eee eer aes pee ents Al Slight. + Slight... 0 0 0
1 Controls.

At the same time that the foregoing test was made a similar one
was conducted with the original nonconcentrated filtrate.

Infected fowls 76, 79, and 82, together with fowls 88 and 89, sup-
posedly noninfected controls, were injected in the right wattle with
0.1 c. c. of nonconcentrated filtrate. All showed a discernible edema
shortly after injection.

On June 23 a slight edema persisted in fowls 76, 79, 82, and 88
(control). On June 24 fowl 88, the control, still showed a slight
edema, fowl 76 showed a slight reaction, while fowls 79 and 82
showed swelling indicative of a fair positive reaction. The results
of these tests and other data are given in Table ITI.

TABLE III.—Nonconcentrated filtrate tested on fowls 26 days after inoculation.

SOP 2p a eS eat See ees ie | =_ —

1 Controls.

TEST FOR BACTERIUM PULLORUM INFECTION IN FOWLS. 5

Comparing the results shown in Tables II and III, it would ap-
pear that the nonconcentrated filtrate has up to date given the most
satisfactory results.

On August 16, 1915, tests of the original nonconcentrated filtrate
were made. Fowls 73, 76, 77, 78, 79, 81, and 82 were injected with
0.1 c. c. in the left wattle. Of these, fowls 73, 81, and 82 showed re-
actions at 48 hours. These had reacted previously, fowl 73 on June
9, and the other two on June 22.

Fowls 76, 77, and 78 did not react from the injection on August 16,
and it is noted that they had not reacted well from the injection on
June 22. At that time fowl 78 was negative and fowls 76 and 77.
were rated as slight. Of four controls, fowls 95, 96, 97, and 98.
injected with filtrate on August 16, one, fowl 95, gave a reaction.

Table IV shows the present test, together with previous and subse-
quent tests on these birds. For convenience the table includes the
results of two succeeding tests on some of these birds, together with
the results of the agglutination test, autopsy, and cultures from
ovaries made at a later date.

TABLE I1V.—Retests of fowls with various test products.

at | 4} fr uo}
e £ S £
pene |e E :
Ooo es g Es =
ss | ea | a oe s= :
Ee on | 2 ce S =
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Fowl] § a | : a S SS
No} oO | Z ‘S 7 A oes
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June June. June \ug. Sept. eas
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SSP ee ee I sale | + - + |+f 4] +
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ee Rae ee eee | ne ee Nines Slight.) Slight.; — | — | Slight.) Slight.) Slight.) Slight. + | + | +
NOs Hea sbaees Seer Seema: ita Soot ji meee aces [eces 245s | Se Slight.| + | + | +
WOR are| = S33 esse) SWOT a= |Sgseeooe Sosasseg eee |) == |RStoceoc | eatstetevers + + se | SF ae
ol 8s Eee ae Cees focSaees J RINE Se) URSE Sse oct oe. eeeecore = 2}—|+
Boe sion ae |_...| Slight. STEN Rca Ps eee abo ee Co | WS tae beeen > Slight.| ? | —}] +
JE bs See Sak eee Sree ea len sdcSqiee= eee FE| Se |lezeagess 2eosa50593sarse5 Socssee: sel S50) =e
POST SSeS Re eee (er pe [<= | am ae (a Oe Oca ae eee =o |aasa ee
TTL LE ein | ees RCo eateries (eee pee ee eine eee hese oe | le ee
TM Ee a a ee a ae eee ee be pe igen Wee eae ened ares ees eee En as }—|—|]—
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1 Controls.

The fact as shown in the tables that three controls, fowls 87, 88,
and 95, gave slight reactions, suggested the idea that an edema per-
sisting for 24 hours or longer after injection might possibly be
induced by causes such as irritation from the carbolic acid in the
test fluid or puncture by the hypodermic needle. In order to deter-
mine this point, 6 fowls were tested, as follows: Two were injected
in the wattle with 0.1 c. c. of plain bouillon, 2 with 0.1 ¢. ¢. of
boullion carbolized to the same degree as the culture filtrate, and 2
received the hypodermic-needle puncture only. No reaction occurred

6 BULLETIN 517, U. S. DEPARTMENT OF AGRICULTURE.

as a result, and there remained the alternative conclusion that the
test was either nonspecific or that the reacting controls were infected.
Some of the control birds had been secured in the open market, and
nothing was known of their previous exposure. Unfortunately con-
trol fowl 87, which showed a slight reaction on June 23, died from
intercurrent causes and in the absence of the authors was not autop-
sied. As appears in autopsy notes later, control fowls 88 and 93,
which also had reacted, were found to be infected birds.

On August 25 fowls 76, 95, and 96 and a control supposedly nega-
tive were killed by bleeding and samples held for agglutination teste.
The ovary of fowl 76 contained angular ova typical of pullorum
infection, that of fowl 96 dried encapsulated ovum and cysts, not
certainly due to pullorum infection, and that of fowl 95 one or two

angular ova and two large cysts.

Cultures were made from ovaries of fowls 76 and 95 which yielded
growths characteristic of Bacterium pullorum, while that from fow]
96 yielded a heavy growth not resembling that of Bacterium pul-
lorum. Fowl 76 gave positive agglutination in 0.01 dilution; fowl
95 in 0.002 dilution, while fowl 96 was negative at 0.04 dilution.

-An antigen was made from the culture obtained from fowl 96 and
tested against the serum of fowl 76, but gave a negative agglutina-
tion at 0.04. The result further strengthens the conclusion that the
infection in the ovary of fowl 96 was not due to Bacterium pullorum.
A check was run on the serum of fowl 76 with Bacillus abortus as
an antigen, with no agglutination resulting. It is seen that fowl 76.
although it did not react well to the intradermal test, gave an agglu-
tination of 1:100. Also, fowl 95 was undoubtedly infected with
Bacterium pullorum.

With a view of securing a diagnostic agent that would increase
the size of the swelling in the wattle, the writers next tried a product
consisting of six strains of B. pullorum which had been grown in
plain bouillon at a temperature of 37.5° C. for one month. The
culture was killed by heating at 60° C. for one hour in a water bath
and then carbolized to 0.5 per cent. The organisms were not removed
from the medium, and this product was employed in all subsequent

tests.
TABLE V.—Tests with killed bouillon cultures.

Edema :
Culture | Aggluti-
Edema after Edema after 24 after 48 } A :
Fowl No.— 3hours, Sept.2.| hours, Sept. 3. hours, |Utopsy. oe pee
Sept. 4. ary: Dee
TN Gad Sess eee Marked ......-. | Marked positive... 5 ee a | + | ++
GFAP a tod a ae Considerable. .| Slight............. Trace..... | at sic ae
IES Wi a ley nee a Shiehtonsneso: | = = 0 | 0 0
|

1 Control.

TESE FOR BACTERIUM PULLORUM INFECTION IN FOWLS. i

On September 2, fowls 73 and 77 and control fowl 186 were in-
jected with 0.1 c. c. As shown in Table V edema was present at
“4 hours in fowls 73 and 77 and entirely absent in control fowl 186.

On September 20 the following birds were injected in the wattle
with 0.1 ¢. c. of the same bacterial product as used in the preceding
test: Fowls 73, 77, 78, 79, 81, 82, and controls 55, 74, 88, 89, 90, 92,
97, 98, and 99. The results of the test are shown in Table VI.

TABLE VI.—Further tests with killed bouillon cultures.

Edema | Edema | ;
| 5s Aggluti-
Fow! No.— te | ou Autopsy.| Culture. | nae
Sept. 21. | Sept. 22. See
he PR OE EE a ea a a fe ate 4. 4 =
TT io Sn yen ope ae RIN rte eae Slight...| Slight...) + a s
Cb) ees 55 es COS ts a CE a Ba a ER + 1 2 alo + -b ao
(OSE TRA Ae ee SET aD Ce Se +) + + + +
SST hos cases a i ln pees PT A er | + leaeeat ? + ae
ROM re aL SRE TN ee Pe Aine MeL Cola et een nS bahia 2 ee at
ile, a ane Re ccs 7 A eae ee a Ne ae re aes ec eaeinees 0 0 Not
| tested
{AG eee er ene ie camer cl eaieienrpehale ue ii aa i er Gee ud = | _ — — _
SO Se eco SaSU eee Baek tenes ies ata ec tae ets ak ee + | Slight... + + +
GOO) TES a a Rts NP eas aie ca) at Soe SSS Ne one RN DR i eee aoe = — = —
OO Eee IE ie ae Swati len! Spe OIC ee I Seas em Ya eat Pe 0 0 Not
| tested.
thes i Fo ri A NO a a alle Re Rs EO _ | — 0 0 0.
Tg) Bh a es Es i SS sie aS a eg Ga + | _ — _ =-

1 Controls.

On September 23, the following fowls were killed by bleeding
and blood was saved for agglutination tests. Autopsy notes follow:

Fowl 73. A hard tumorlike mass about an inch in diameter is attached to
the ovary by fibrous threads and vessels. Other small ova show typical ap-
pearance of pullorum infection. Bacterium pullorum was recovered in pure
culture from the ovary.

Fowl 77. Ovary contains one ovum the size of a pea, having the appearance
of an old pullorum ovum. B. pulloruny was obtained in pure culture.

Fowl 78. One ovum the size of a pea and having characteristic color of those
With pullorum infection. Also several smaller similar ones and a cyst the size
of a hickery nut which is filled with a colorless fluid. B. pullorum was obtained
in pure culture from the ovum.

Fowl 79. Ovary contains a number of typical pullorum ova. Pure culture
of B. pullorum was obtained.

Fowl 81. Ovary contains large ova apparently normal. There are some
small brownish ova that may or may not be infected. Liver contains a few
necrotic spots. Cultures from both liver and ovum gave negtive results.

Fowl 82. Large ova apparently normal. Some small brownish ova that may
or may not be infected. Liver contains a few whitish spots. Cultures from
both liver and ova gave negative results.

On September 24, the following control birds were killed by
bleeding, and serum was saved for agglutination tests: Fowls 86,
74, 88, 89, 97, 98, and 99. All were normal, and cultures were nega-
tive except for fowl 88, which had reacted to the wattle test. This

3) BULLETIN 517, U. S. DEPARTMENT OF AGRICULTURE.

bird was found to possess a typical pullorum ovary, and yielded a
pure culture of Bacterium pullorum.

Tn preparation for another set of trials of the test upon artificially
infected fowls, on August 26, 1915, the following birds received
intravenous injections of 1 c. c. of bouillon culture of nine strains
of B. pullorwm mixed: Fowls 28, 67, 85, 182, 294, 204, and 215.
On September 7 the following birds were injected intraabdomi-
nally with 5 c. c. of a similar mixture of strains: Fowls 16, 18,
82, 34, 36, 87, 88, 39, 45, 46, 47, 48, 49, 64, 65, 69, 76, 178, 190, and
197. Fowls 28, 85, and 215 died shortly after the injection.

The birds were tested with killed bacterial culture. The results
of the two tests, with autopsy findings and results of cultures inocu-
lated from the ovaries, are given in Table VII.

TABLE VII.—Two fests with nonconcentrated hilled culture.

Fowl No.— Dec. 7-8. Feb. 9-10. Autopsy Mar. 16. | cue

AL Geir nee eter coh eres et ecreantckayareie ae fers + -- + = Questionable. . . | ze

SS eee Ne anes SL veh noe Sysio es eye rcleinta niet) eras | - — — — Rositivieneeeeee ame

UN's Sat a a deli osm elas pe gD Se a Rage feats + aii donc ees at

SYN gr cea SR CE MUI or gt BRR REN erga Rd ee ee Meg oto | — = Trace ie | doe eae at

Gee ele Ga nee tele g te OR ge ct ahah da pall eeymiact au ar Trace _ Questionable...{ —

seat eae Mercere Bi peso coos Pen nye gat a + Srey lees GOs. Goss =

ey Birth t cenl i ines Mar Galea Ne tad Ned Pong Nf et mmapeter eS em Veet ry erst + Trace IPOSIbIVIC;Seee ee gous

BYR) EEG gk, SA ari aenigth sae al pn ae ee ee oe Je = Normal: sane ese e

A Reis Soe tes frees et So ne Sree a eal aber vats Steieye rete iaieiai |} + at + SG ese GOs Sore =

ZN GY ae oe Ae a ae ae eS eta a cs UO Ete a aie Trace == tia Ae dort Ea eee =

dG ASE i NTs he AO Ui vo vcard OR aan lites erat Ge a po +. | + + + IPOSILIVElse eee =

NAS ih OATES wns A Sa A ee OSORNO hopes ae = — Normals see a3
AOS Mt ae Deere se HNN cc 4 dela a pene | ayaa dl eee |e REACe — Questionable...) —
(RNS Sa veh tesa nok) RAEN aa MEd UR br [te au + — POSitiviexs ---s2-e =

Gee em rE eae Ay Me anten eed cast meu + = — a Normals ma

(Tee. 1 An ae cone Ree Bena RRO, RAC Rs cae at gee a ak aL Trace — Positive:-245-2- =

(BO) art SON ctr eh de a en re dae opr ONS tn AR |} ae = + _ Questionable....| +
INR a eas el ao Sh mae en Me as eR + + + _ JOOS Osoce5455¢ Be
TUT aes esse eel aoc Gee SSeS Se gat Ueno ERO ee | =e = | + TACO) esos e doy eee =
TUCO ee NR ee eR Ca Neate ry Gk am Nc + af) | + Draces||eases dome ee _
197 we et et ee rt tee et ete ee ee eee ee ee ee eee + Fr soa ae ee ekanevets: do a =x
TSI Sits ty ile ee baa Pe ne Nn See eS RNR Sn + + + Race nal eee dios Sees =
DNA Ni ays ae tis seat AUC Se OL ent Nd sepa Mi Reiki te + + Trace Questionable ==
SAO Sale ole et Cem SM Senay eee eM eee ae Re rhe eects — | Trace. = Normal i= =a. —

Ai lemaeemne Rtces Merete neck co oie amr eaea aE ee Ee ase TH — =| _ — Not aalled worn alaoee ere
AD UES Fae ore Se ere af ekss arate) Sector een cs is Mae a — —— >| _ se A Se OR aes Rees
EL ey RES RE RRO et NES ens La HE EOE DN — — | _ = Norm alse Se | emeeoeer:
Dye SA gees ae ae Cai ee ig ateip amt eat oh Ne | ot — | = = Died: <. -senaees _
Tg ee eee See ee Se cera eel seen sera cee -- — | = = Normal (2 2253-26) err
SSA) 5 as Ores eee SAL oy one eee ann EOPUan ee Lt ae = = | — — Not dalle? Pea eee pune St
TIPSSTLs Misc 2 a Rec Pac ae ape trans Nezs aM en a OR = = = a Se Setece OWS eee ae aoe
1 ole CeO eno ees Hine se cia ye le -- — _ > Eee aa oe OX... oes |S
FL) RU Te MNS eye avs Stream ee ets RE oe BN ee — | = — =| earl d0:s ashes tee
TT eS eee cre Coa oe LS LIN a ate a aca yan i — — _ Soo eer GOs 2 Bee ee

1 Controls.
SUMMARY OF THE TESTS WITH ARTIFICIALLY INFECTED BIRDS.

In the course of the experiments recorded in the foregoing, 32
birds that had been exposed to infection by injection of live cul-
tures were employed. When tested for the first time 29 of these, or
90 per cent, revealed edematous swellings rated as either slight or
positive at 24 hours after injection with the diagnostic agent. When
read at a 48-hour interval, 23, or 71 per cent, of the same birds gave

TEST FOR BACTERIUM PULLORUM INFECTION IN FOWLS. 9

reactions rated as either slight or positive. Thus, the 24-hour in-
terval yielded the largest percentage of reactions. Practically all

~ birds, both those inoculated and controls, exhibited a swelling shortly

after injection and therefore no diagnostic value has been attributed
to swellings observed before the lapse of 24 hours.

Three birds gave negative readings at both 24 and 48 hours.
Autopsy of two of these revealed unquestionable lesions of pullorum
infection, from which the organisms were obtained, while in the
third one the lesions were questionable and no culture was obtained.
Thus, the test failed to detect 6 per cent of the birds in which lesions
were found.

In all but two cases the same birds were retested after an interval
of 7 or 8 weeks. Of the 30 birds retested 22, or 73 per cent, gave a
reaction rated as either a trace, slight, or positive at 24 hours on the
second test. At 48 hours on the second test only 8, or 26 per cent,
displayed reactions rated as a trace, slight, or positive. Further, 8
birds, or 26 per cent, showed no reaction at either 24 or 48 hours.
It is evident that a retest after an interval of about 8 weeks is far
less reliable: than a first test.

Of the 32 birds tested, autopsy revealed unquestionable lesions in
18, or 56 per cent. In 8, or 25 per cent, the lesions were regarded as
questionable. In 6 birds, or 16 per cent, no lesions were found,
although all were positive to the test at 24 hours.

Twenty-six controls were tested for the first time. These had
been gathered from various sources and there was no assurance that
they were free from infection. Of these, 5 at 24 hours after injec-
tion displayed swellings rated as slight or positive and 4 displayed
the same condition at 48 hours. At autopsy 2 were found to be in-
fected, and 1 through accident was not examined. No lesions were
found at autopsy of 2; however, 1 of these came from the same flock
as one of the unquestionably infected controls, and had been in the
same cage as the infected bird.

While agglutination tests were made on serum drawn from inocu-
lated birds, after injection with the diagnostic agent, and the results
appear in the various tables, it 1s realized that agglutination would
naturally be expected as a result of the various injections. We have
observed that as a result of the artificial infection with cultures of
Bacterium pullorum, the agglutinating value of the serum of these
birds varied within a wide range. Some birds gave an agglutina-
tion at a dilution of 1:1,000, while others that had been repeatedly

‘Injected with the test fluid gave no agglutination, owing to the strong

bacteriolytic properties of their sera, presumably resulting from the
various injections. Negative control birds after one injection with
the test fluid gave an agglutination titer of 1:50.

10 BULLETIN 517, U. §. DEPARTMENT OF AGRICULTURE.

The disadvantages of work with artificially infected birds, due tc
the large amounts of culture injected and to the severe reactions re-
sulting, were thoroughly realized, and work with naturally infected
birds was undertaken.

FIELD TRIALS OF THE INTRADERMAL TEST.

Through the courtesy of the Connecticut agricultural experiment
station, opportunity was afforded to apply the intradermal test to
two flocks tested at the same time by Dr. L. F. Rettger by the agglu-
tination method.

One flock of 231 birds injected on February 28, 1916, contained at
the time over 40 birds showing more or less evidence of swelling of
the wattles due to frostbite, while 6 others showed very slight swell-
ing attributed to the same cause. When examined 38 hours aiter
injection none was regarded as showing reaction to the intradermal
test. One bird gave a reaction to the agglutination test and was
killed by the owner before arrangements were made to retest by the
intradermal methcd. However, the owner had made an autopsy and
reported that he regarded the bird as infected.

In the second flock in which work was done the Connecticut Agri-
cultural Experiment Station tested 50 birds in the regular routine
work of testing. Of these 1 reacted to the agglutination test and
failed to react to the intradermal test when examined 46 hours after
injection. A number of birds showed slight abnormal conditions,
regarded at the time as due to frostbite, but noted in connection with
the problem of determining the least amount of swelling to be re-
garded as a significant intradermal reaction, under the conditions in
question. :

The bird that gave a positive reaction to the agglutination test was
retested by both methods about a month later by Dr. Rettger. At 24
hours after injection the wattle was swollen to about 2.5 times normal
thickness, and when observed at 48 hours the swelling was 1.5 times
normal. An agglutination test made at the same time also gave posi-
tive results. It is probable that the failure of the intradermal test
when used the first time was due to some error in technique. Further,
it is the belief of the writers that readings should be taken at about
24 hours, and not as late as 36 and 48 hours, as in these trials.

In the same flock the intradermal test alone was applied to about
100 birds, and those showing any enlargement of the wattle at 46
hours were tested by the agglutination method by Dr. Rettger. The
results yielded by both methods are given in Table VIII. The size
of the swelling following the intradermal injection is indicated as
nearly as possible by arranging them in order of decreasing size from
the top to the bottom of the list. Here, again, cognizance was taken

TEST FOR BACTERIUM PULLORUM INFECTION IN FOWLS. 11

of every degree of swelling, without implying that the slighter
swellings were significant.

TABLE VII.—Comparison of intradermal and agglutination tests.

l
| Agglu- Agelu-

Fowl i ‘ tina- || Fowl , é tina-
Nee Intradermal] test. ment || Noe Intradermal test. er

test. i test.

| 66 | Swelling and drooping of feathered

93 | Whole wattle swollen, <3; droops..,
skin at edge of wattle. .......... --

76 | Whole wattle swollen, x3------..-.
77 | Lower half swollen, X3-.......-----
8) 1) SwOllad, Seo 8 seo on cedkecass WELLS ek ee eit EE aes se IN -
72 Swollen, SAO) SAL el AMER Ra oe Swelling possibly due to trauma-
96 | Sw ollen, SOL BUS Saar tism, as wattle is very blue..._.. —
99 | Lower half swollen, Sp! SURE RC re 60 | Questionable swelling of feathered

=
3
—,
le)
Oo

59 | Lower half sw ollen, SIM SIAR at Ban a — skin at edge of wattle suns | 2+
81 | Lower half swollen, CI Ones) { — 95 | Trace of swelling on posterior half
100 | Lower haif swollen, X1.5.......... = Olwabllennte ite hee ia ea —

97 | Lower halfswollen, X1.5..........

The results were particularly discordant in the case of fowl 93,
which had been placed at the top of the list as showing the best
intradermal reaction, while it failed to give a reaction to the agglu-
tination test. In view of the discrepancy, Dr. Rettger obtained the
bird in question, together with three others, for retest and autopsy.
The results are shown in Table LX:

TABLE IX.—Comparison of retests and autopsy findings.

Intradermal test. Conditi
Fowl Agelutina- ondition of
Ais ovaries at
No.— tion test. antGnce
24 hours. 48 hours. CABO USB ic
CB ea SV Ulead a ha Sees, = = Normal,
Dee Tes Swollen, SOD raya Swollen 1.5.._. = | Do.
DOs — — — ' Normal (small).
60a: — | = a | Normal.

| | |

The result of the retest and autopsy of birds 93 and 72 is not
wholly satisfactory. The repetition of the reaction in both cases is
significant; but, on the other hand, the results of the agglutination
test and autopsy leaves the matter inconclusive. As to the remain-
ing discrepancies in Table VIII, the many other cases noted as surely
the result of freezing indicate that it is not desirable to apply the
intradermal test where there is a possibility of freezing.

TRIALS BY OTHER INDIVIDUALS.

In several instances the test product was sent to interested indi-
viduals on request. One report on the results was received in which
1,301 birds were tested and 78 gave a positive reaction. The latter
were retested by the agglutination method, ane 70 gave a positive
reaction.

12 BULLETIN 517, U. S. DEPARTMENT OF AGRICULTURE.

COMPARISON OF RESULTS OF AGGLUTINATION AND INTRA-
DERMAL TESTS ON NATURALLY INFECTED BIRDS.

Through the assistance of Roy E. Jones, we located and purchased
47 birds that had given positive or questionable agglutination tests,
applied by the Connecticut Agricultural Experiment Station. These,
together with nine controls, were injected for the intradermal test
on June 23, 1916, and readings were taken at 24 and 48 hours.

Of the birds reported positive to the agglutination test applied by
the Connecticut station, there was total agreement in 28, or 70 per
cent, of the cases in that they also gave positive intradermal test as
determined 24 hours after injection and displayed unquestionable
lesions when eventually slaughtered. Of those reported positive to
the agglutination test, 30, or 75 per cent, revealed lesions at autopsy.

Thirty-five birds gave positive reactions to the intradermal test.
Autopsy revealed that of these 29, or 83 per cent, possessed un-
doubted lesions, in 5 the lesions were questionable, and in 1 no lesions
occurred. Of those reported positive to the agglutination test, 3
birds, or 7 per cent, failed to react to the intradermal test, and
autopsy revealed no lesions. On the other hand, 2 birds, or 5 per
cent, that had given positive agglutination tests, gave negative in-
tradermal tests, and autopsy revealed lesions. Thus, the percentage
of absolute failures of each test as judged by the other test and by
the autopsy findings were very similar in amount.

Seven birds had given questionable agglutination tests. Of these,
3 were negative to the intradermal test and negative at autopsy.
One reported questionable gave a positive intradermal reaction and
autopsy revealed lesions. The intradermal test on the other 3 yielded
positive, negative, and questionable results, respectively, and autopsy
of all 3 furnished inconclusive information.

Of the nine controls, one displayed a marked reaction at 24 hours,
consisting of a swelling of the wattle to three times its normal thick-
ness. Autopsy revealed undoubted lesions, and a pure culture of
Bacterium pullorum was isolated from the ovary. Four others dis-
played traces consisting of swelling of the lower border of the wattle
to about twice the normal thickness. On autopsy, one of these was
found to contain undoubted lesions and a pure cuiture of B. pullorum
was obtained.

The examination of the wattles at 48 hours revealed swellings vary-
ing from a trace to positive in only 22 birds, or 46 per cent, of those
tested. This result compared with the 28 birds regarded as positive
at 24 hours and verified by subsequent autopsy, again indicates that
48 hours is too long to secure all the positive reactions. Among the
controls only 1 displayed any swelling whatsoever, and this case
proved on autopsy to be positive.

TEST FOR BACTERIUM PULLORUM INFECTION IN FOWLS. 138

On June 26 all the 47 birds and 9 controls were reinjected and ex-
amined 5 hours later. At this time every bird, including controls,
displayed a swelling varying in the different individuals from a
trace tq five times the normal thickness. The observation merely em-
phasizes the fact of the occurrence of a nonsignificant swelling fol-
lowing injection with the diagnostic agent.

At 24 hours 39 birds displayed swelling of the wattle varying from
a trace to enlargement to five times the normal thickness. Autopsy
revealed undoubted lesions in 380 of-these, questionable lesions in
7, and no lesions in 2. Total agreement between the results of this
reading, the agglutination test, and autopsy findings occurred in 70
per cent of the birds tested. In two cases, or 4 per cent of the
_ birds, the positive readings by the agglutination test were not sup-
ported by the negative results of the intradermal test and the
autopsy. In 1 case, or 2 per cent, negative results of the intradermal
test were contradicted by the positive results of agglutination test
and autopsy. Thus, the results yielded by the first and second 24-
hour readings of the test on supposedly infected birds vary but
little. |

The results yielded by the test on the control birds were perfect,
as confirmed by the autopsy. The only two birds that displayed
traces of swelling proved on autopsy to be infected.

The fact that the results of the agglutination test, intradermal
test, and autopsy are in complete agreement in 70 per cent of the
cases, coupled with the fact that the absolute diagreements are very
smali, indicates that the two tests are equally accurate.

The results obtained at the autopsy of the birds emphasize the
difficulty of determining a standard for comparison of the accuracy
of the two tests under trial. Thirty-one cases, or 64 per cent, were
found to possess unquestionable lesions consisting of the angular ova
characteristic of the infection. All of the cases had given positive
reactions to one or both tests. In nine cases, or 10 per cent, the
autopsy was inconclusive in that there were present only very small
dark ova or cysts. Of these 9 questionable cases 3 had given ques-
tionable agglutination readings but positive intradermal reactions.
In two cases the agglutination and intradermal tests disagreed. In
four cases both tests had given positive results. 3

SIGNIFICANCE OF SWELLING AS AN INDICATION OF A REACTION.

In determining the significance in diagnosis of an edematous
swelling of a wattle one is confronted with the fact that in all birds
such swelling occurs shortly after injection. The problem is to
determine the point of time after injection to read the test when
this preliminary swelling has disappeared, yet not too late to escape

14 BULLETIN 517, U. S. DEPARTMENT OF AGRICULTURE.

observing a significant edema. ‘The tests on birds in the laboratory
and probably also those in the field indicate that 48 hours is too late.
While some observations on birds in the field made during freezing
weather would indicate that slight swellings should not be con-
sidered, yet the entire experience with birds in the laboratory indi-
cates that even a trace may be indicative of a positive reaction. Some
few cases would indicate that a 24-hour reading might give false
results due to the inclusion of some cases in which the preliminary
nonsignificant swelling had not quite subsided. At present, the 24-
hour interval has given the best results, but the examination of a
series of readings at 30 hours would be desirable.

VARIOUS BIOLOGIC TESTS.

During the course of these experiments several attempts. were made
to produce a reaction to the diagnostic agent by injecticn into the
comb, but no satisfactory results were obtained. The ophthalmic,
palpebral, and subcutaneous tests also failed to produce a reaction.
Also limited complement-fixation tests on the blcod serum of infected
fowls gave uncertain readings.

SUMMARY AND CONCLUSIONS.

A killed culture of Bacterium pullorum grown for about a month
and held for several weeks before use and without further treatment
other than carbolizing, has given the most satisfactory results.

It seems to be a fact that the edematous swelling resulting from
the injecticn of this product into the wattle of a fowl, when observed
at a proper time interval, is an indication of the presence of infection
of B. pullorum in the fowl.

Our experience to date with readings at various time intervals leads
to the conclusion that the 24-hour interval has given the most accu-
rate results. However, it seems desirable to test on a large number
of birds the accuracy of readings made at a slightly longer interval.

The weight of evidence indicates that any perceptible swelling of
the wattle should be regarded as significant. A second intradermal
test made at an interval of four days gave results varying but little
from the first test. Others made at intervals up to two months gave
less accurate results the second time. Thus, there is no advantage in
retesting.

Of birds artificially infected with the disease and tested in the
laboratory, in round numbers 90 per cent gave positive reactions; and
in 6 per cent the test failed to indicate a reaction when lesions were
present. In 3 per cent no reaction occurred and no lesions were
present.

TEST FOR BACTERIUM PULLORUM INFECTION IN FOWLS. 15

In a field test'on 231 birds made simultaneously with the agglutina-
tion test, the intradermal test at 38 hours failed to detect one case
reported positive to the other test. In a second flock of 50 birds in
which the two tests were compared, the intradermal test when read
at 46 hours failed to indicate one case that was detected by the agglu-
tination test. Another group of about 100 birds tested under un-
favorable conditions gave less satisfactory results.

Forty-seven birds that had been tested by the agglutination method
by the Connecticut Agricultural Experiment Station in the field were
purchased for experiments with the intradermal test. Of these, 40
had given positive reactions to the agglutination test and 7 doubtful
reactions. There was complete agreement between the agglutination
test, the intradermal test, and autopsy findings in 70 per cent of the
eases. The agglutination test reported positive in 3 cases, or 7 per
cent, was not confirmed by the intradermal test nor by the autopsy
findings. The result of the intradermal test was negative in 2 cases,
or 5 per cent, when it was not confirmed by the positive agglutination
test and autopsy findings. Thus the percentage of absolute failures
cf each test was small and very similar for both tests.

Autopsy does not furnish an absolute standard for comparing the
accuracy of tests. Seventy-two per cent only of naturally infected
birds that had reacted to one oer both tests were found on autopsy to
be unmistakably infected.

The intradermal test detected the presence of infection in 4 of the
34 control birds injected in connection with the tests in the laboratory
on artificially and naturally infected birds.

In a field trial not made by the writers, 1,301 birds were tested
intradermally and 78 reacted. Of these 70 reacted to the agglutina-
tion tests made subsequently.

The intradermal test has already shown sufficient promise to war-
rant further extensive trials in the field in comparison with the
agglutination test.

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WASHINGTON, D. C.

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