The cause of European foul brood

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Title:
The cause of European foul brood
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White, G. F ( Gershom Franklin ), b. 1875
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Government Printing Office ( Washington )
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Table of Contents
    Front Cover
        Page i
        Page ii
    Historical and some earlier work by the writer on European foul brood
        Page 1
        Page 2
    Recent work by the writer
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
    Probable explanations of errors as to the exciting cause of European foul brood
        Page 12
    Summary and conclusions
        Page 13
        Page 14
        Page 15
        Page 16
    Back Cover
        Page 17
        Page 18
Full Text
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L. 0. IIOWARI)D, E it,,,vIlgil 1i1dl thi f ,if Il-'i iut.
C. L. MARI IT-. I"ln t1,t',ll'lgii i lnil .A( ling ('i irf in .Ib\ iv, of 'Ihirf.
It. S. (CLIFTON. .t i filliri 1 i.,i'l riil.
W F. TA.-II r. 1', itf l' I ..

F. H. CiI ITTMENIE.N. in charge of truck e-roi, ,l .xluor'd Inoilurli in.u' ii ,' .l iatifiions.
A. D. HorKI NS, in charge of forest iiinvl ii rv'sliguitiuOn..
W. I). HIUNTER, in charge of southern firlih (ripIi in.rcl itn .''liiljlit,,.
F. M. WEBSTE, in charge of cereal andl fuirigr" intfscCe ili i .%liili'iui'.
A. L. Qi.i NTANCE, in. ciimpt of decidluouii. fruit insect in 'rrfigifin,.
E. F. PIILI IPS. in charge of bir culture.
D. M. ROGERS, in charge of re I'i tliu .re 'iinl oif nt/,.., flild uri'i'l,.
ROLIA P. ('TURiti in inrieiir of editor i'll wirl,.
MIABEL (COLCORD, in ihii if' of lilt,'iil.

INVESTIGATION.N IN lit.I. '(I TIRE.

F. F. I'111[I.i .,s. in vliirg '.

G. F. WHITE, J. A. Nt I.SON, eXl0u'rl.
G. S. DEMUTII, A. H. McCHAY, N. E. M(ItmNiH. alpnullurt u.i..xjantis.
PEARLE H. ;.\HHI:'sN. ipF'l ii'i.'
H. A. SURFACE, D). B. CASTEEL. tollitbiio'iis.
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CIRCULAR No 157. Iudit M 12i.

United States Department of Agriculture,

BUREAU OF ENTOMOLOGY.
L. 0. HOWARD. Fniormn,.lov.it and Chief of Bureau.


THIE (CA UIS: OF FlUIPEAN FO ,L 1I911OI).

By (I. F. \ NHITS:, M. ID., IPlh. ID..
I'rl ri rt in /*'*'*t,,;'," ,I'-

I ISTORIi'AL.
Tliw plrpo!- ,,f this prelimninary paper is to disciis, Inrifl\ the, ex-
(itili l.ui-rv of European iiil brood.
With cli.i'lI'ed material fiirnis-hed by ('l.-I ir., ('liiir'i made a
bacteriological .itidy of foul brood. 'IT latter i-,0l ii I and described
ii bacteriumn from the brood dead of the disease and identified it as
Bi,, ;'lhux ul,. i. ( 'h -li ire :iiret',l that the ii liil iti ilioni was correct.
A joint paper lbv ihese men appeared in ll.",. and for about Ia ,ih-adiil
and a half there:t'lter BJ ill)u, al'ei/ was L.incrailly considered to ibe
the cCAuSe of fillil brood. The disease whibh. it is b1ileved. Client.vi
st tudlied is the oin.e that has received the de.ignition European foul
brood.
Thi.- dlis.ea-e is iilso believedl to be the one which \Villinm R. IIov:ird
worked upo in and ii:i nie '" black brood.n" In P10'1 he de-,cril,''d as
it- t'aiii-' i:ibctcriilin to which ho' ir:ivt the name ltn,.;ll/. mrlii.
Ii is priobiable 1it:it Burri in 1101; wa.s -.riyiti i the same disease in
Sw itzerlind when he rc frrrt.I to" -mour brood." In the disease he li'.
cIv 'er0 It baicteriumn to %l which li, referred as the 'p/0tolu '/--frinl-."
Mii-en n I 907., \vorkill probably with the llme disease. .i 1p11:11i-
Ol lV t'liCi' 'i il 'I'I the ", ,,,/ ,, r;-fi'ii reported lb Burri and
Ilillli d thi Il '\'w pe'ii'-Sf/,, ,tiv . ".' '#1';'.
(Mi lF : %R. IFR 'UtKK BY TIlE. WRITER ON EUROPEAN FOUl BR(iOD.
Tin I.'ii7 (he writer il'-'rviled in Eiirupean f,,,1l brood tihe bacterium
wVhih hliad It ,TII named Shtptoroct.N aitps. It was i:i-,rvel at lie
bailey t i lt thailt there w preent alo ;inot lIer iiii 'r ,i.,'ni -in quite
1






THE CAUSE OF EUROPEAN FOUL BROOD.


similar in appearance, but clearly different from the one which Burri
had observed and Maa-,seii had named. All attempts', however, to
cultivate this new species were unsuccessful. Ustil more was known
about this org-li-ni it was referred to in 1908 as "" B, ;1is 1'."
In 1907 the writer demonstrated that American foul brood, an
infectious brood disease, could be produced by feeding to healthy
colonies pure cultures of Bacillus larva. This fact emphasized the
probability that if European foul brood i al.o caused by a bac-
terium this (lis-.ea e. too. could be produced Iby feeding pure cultures
of the bacterium ca using it.
To demonstrate this, it was desirable in the first place to determine
whether or not the virus of European foul brood was present and
active in the diseased brood. Healthy liee" were fed sirup which
contained a suspension of the diseased brood, and European foul
brood was produced. This showed that thle diseased br-ooil did con-
tain the virus and that the disease couhi lie produced by feeding.
This being done, pure ult uii-e, of Bacil,,, i. ,,. isolated from the dis-
eased material were subsLituted for the livase(d brood il l he inocu-
lation experiment. Pure cultures of St, ,'/oocct.s api.s were isolated
and used likewise. Then cultures of lfib, ; -,,,. il', ;nal iulture of
Streptoocccus apis were used simultaneu-lv in making the inocula-
tions. European foul brood was not proilu.ed, in any of thlie experi-
ments where pure cultures of either or both organi-um- were used.
These facts were sitlicient to eliminate tentatively Ri'i,, ;,1,., ai',-i and
Streptococcus apis from the list of po-ible c.l-es of European foul
brood, and to justify a strong s -Ipicitin that the microorganis m which
was referred to as BiIlbs Y bore a cai' .il relation to thlie disease.
It was necessary, however, to reckon with other factors before a
more definite statement could be made.
A continuation of the work on the c:lIu-e of Euiropean foul brood
lij.l-, yielded some intere-ting results. These will lie b)rieflv con-
sidered in this preliminary paper.
It has been observed in the examination of dlicea-ed brood that
Bacillus alvei is frequently either absent. or pre-,ent monly in small
numbers, in many of the larvae which seeing from gro,- appearance to
be dead of European foul brood. Such -a mlple- liive been received
as a rule from localities in which apparently thIe disea-e had only
recently appeared. Frequently, Amlo. ', ,Iclto ,i,' i, ,/ei., seemed to
be ahbsent, or present only in small nimibers, in many of the larvae
which from arons appearance Lrave strong evidence e of European foul
brood. These bacteriological findings further strengthened the
theory that neither Bacillus alvei nor Strcnpto,'ouu'.s 'f>'s i- the pri-
mary exciting cause of European foul brood. Oilier inoculation
experiments were performed, using pure cultures of tlme-e two species.








T IIe rti Ilt w rr always t ,...Iillve,, ":iliII furIlli r -,iIIil.,r r, -Ill,,
tlintl 1 iiil Ibeen ir(vioIuslv obtained.
|IE( i~r \\(lli lIy TIll : W1SITKI.

W1 iile h11 -I II .'t were in I \neasurIe -IiI- f :*t' ,rtv, as IrI, Iir,-
I rtro, / vly tI I i i 'IicatIe certain i c nclusio s-, there was still tli,.
lint h',Lrrv',. t ,of ,.unclusivne'ss which is always dhvsiredl. liy eXlpri-
int, iiio i iLt i Ti t I the study ,f the brood s )ick or IIead of ithe
(lis' l;i-, tli 'li \as i i'il, 1.1,ll [v ',,i .li I. hIwI' aer, 1 .oiii-tLraldle 1ii-
f i'm-I tion ol' ( lie character IlTi. I for I;Is ien itaIined. 'Ilir.
'let'ail. 4,rI tIw .lm h 1niqiie used in II ,11I liiI t 1h inO ,cula-tion ill nlt Ic

i t i, l i I. lr l- lf rI I r I. i
K XIEZlIMESTAI. INOCM'l'ATIlON

I)i-,''i.ei ni~itrial frm eriiair s lIoalities \a\n used for tlhe-t inwcu-
lhiti,-. Sn,', 'olonic- were fed diseased bro I tIhat conta iiced, a-.
re~peale, I Iv n-illtral examinations, l.,uur, quantities ,,f Bi/ifillns other colonic. were ftl diseased material _,tl:i Iuiiii 1:1 n., nu*nr lr
of Mf,'1,/I., IT ;,. I,,i,.- as shown by ii titir-. imn still others were
feil di..eaed niimaterial which was demonstrated to contain neither
Bit -!f, s ,.,fi i' ,r tf mw il "cus8 ai/ps. It was fi6uiiiI that at the fir-st
Ilp|IKil i'ncIT ,Id lie li.-aease in each class of experiments the symptoms
nmi nfec.t cr I'v thie sick larva' were the same. Larva: shlowin these
c rly -vi p ,toi, were studied bacteri''7it ly1. rin, examinations
shmwei tlhat whether or not the di-.da-,I material fed to the bIeeI
contained RBa'ed,,s alrt; or S.tri ,/,.ni r,.. atps these species were
in the early st..ges of the diseas-e either absent, or present in small
miinmer. only. IT is quite evident that the disease was not pro-
duced bw siWies of bacteria which were absent at this early -t,.ir'
of the diie:se.
Continintilir IlI bIacterio1iogical -tuiidy Iof (lthe larvw in the e.irlv
sta-'r- of lthe li-.ei-. some new species were foundl to be present.
(hne h:mtierIim u-. ,ecially is frequently encountered. TIi. sp)ecie-
is a small, slender rod. apparently nonmotile and non-pore IcAbin,_.
It is t, I w knivn iV the name fuii, ', r1,,in 0 ,,.il;, ,. Its, de-cription
will appear in a later publication. '. \xlriimnental colonies have been
fed pu1e cult tIre- i f this -p,',ie-.. lht no disease has Ieeln 1,rI,,li'-,1.
Tent: t ivel v. tiiref,,r., this spe'it.- is not to be reL'iridt'led as the cause
of Eiir'pean foull brood.
Two milther -,cri,- (if Im:icria mighlit lbe mentioned ;,'I as IliI._
of intere-t in connection with the study of thle broos! dieases. The
ir-t to le millentioned is a iiitile. vare-lI ,riin .. e;i-ilv niliv.'t;lailil.
rod. It i- to re,,iv' the name B ,l', 'll,. .,ii,, ,qi. Tt als'o %ill be dv-
-cribed later. This -pecie-. is ,,.c-i,,ini:lly found in very lr!rt' 1 nuim-


T illm ; t' it., n I.1 liw .\ tIi l'il'l, Ill ir ii i.






THE CAUSE OF EU'ROPEANX FOUL BROOD.


ber- in .-ampiiles of Eiir,,pean foul brood. Feeding it in pure cultures
has so far given negative results. This organi-mi. then. can also
be eliminated tentatively from tlhe list (if pos.il-ile cause.- of this
disease. The other lpwcies. mentioned by Lanixiitte in 1902 as
Bacillus vmesentedicwu vdl/ifir;.S. may l)be -aidii to belong to a .roup of
bacteria found quite widely i .tribute(-d in the :lpiarv. Its infre-
quency in diseased brood and its occurrence in small iiinnhers readily
eliminates this species from the li-t of pos-ible cause,. Most of the
bacteria that are met with in the study of European foul brood were
therefore excluded tentatively from the likt of possible causes of the
disease.
The possibility of an ultranimicroscopl)ic virus. was al-o considered.
Brood sick or dead of European foul brood were removed from the
combs and crushed. An aqueous -ui-.penion of this diseased material
was then made in boiled water and(] filtered with the Berkefeld filter.
The firtrate remained clear when incubated at different temperatures
and cultures made from it produced] no growth. Separate fitrations
have been made of diseased brood recei red from va rious local it ies. but
in no instance where healthy colonies were fed filtrate obtained in
this way was European foul brood produced. The results of the ex-
periments therefore justify the tentative conclusion that there is no
filterable virus in European foul brood capable of producing the
disease. To this extent, then, has the possibility of an ultramicro-
scopic virus been eliminated.
Having thus tentatively eliminated all the microscopically visible
organisms except Bacillus Y from the list of possible causes and like-
wise eliminated the probability of an ultramicroscopic virus, the
tentative conclusion was naturally reached that this remaining micro-
organi-m probably plays an important rile in the etiology of Euro-
pean foul brood. Such a conclusion was all the more imperative
since this organism had been encountered so frequently in the brood
of this disease and since, moreover, there had been no other factor
observed to which the exciting cau-e (ourld be attributed.
SYMPTOMS MANIFESTED BY LABRV.: qIt K OF EVROPEAN FUL BROOD.
This conclusion led to a more extended studl, of this microorganism
in the disease produced experimentally. Thlie pre-ence of disease
can usually be detected in the experimental colony during the week
that the feeding is begun. The first indication of it. may be that
only a portion of a larva is seen in a cell fig. 1). the remaining por-
tion having been removed by the bees. A-,ide fromni an observation
of this kind the earliest indication one get- from the microscopic
(gross) examination is that sick larvw are found among the uncapped
brood. One should acquaint himself, therefore, with certain symp-
toms or signs manifested by sick larva, during the course of the dis-












2 -.


Ft.: 1 I .nri> 1 -hi ,,f I:'ir..I. -In f Il I'i,. 2 ll ,ilth Iilrn a of th. age reprr-
h!'.r..'..l i.iTIi r. in.%.. '1 I <. hflt In f r" 4. i Lr.Ig nal I
i i iri.liiual
Thel Ingtlli of ti i tllit a1 l','h1iL"L beelw i -Wk o1f Fturopean foul
brood i'. variablet. It -.n 1I e -t.;tt,.1 in ai pg i'J r.i way that the three
1f i ii ,/*''


Fin; Sick larva r.f th' Ii:," rt'lr. -* I'*
In flLirrc- nn'1 4i i4 'rq1 .inil
lnv'"- jn..t prfin,',lini.'l-" '1 iu* ,,,h, n
it liirva %\oiill t ,rli-< ilv I,. >.il ,,.,]
i, thlie inim st f: ,r:il,'- l,'n i' ,l tf'r
nmakingi: ni dliign,-i- frni the .'r,--
exainiin:atiTin alnt'.
When healthy I lrv.r ,,f the .,'g'
represented in tiirmii- 2. :3. 4, and
5 are slightly magnified a peristal-


Fir, 4.-I ck lana with rof of I 1ll
moved. Iriinal i


ll': <.'.\ '.l' l I 1 1 1: %' N I I I I. In" 1 .


. -,'.. bv" Il l, 1 it- i~n -r' ,,' .11l IN. ,1i .iL ...", w hill- tlit 0.Ir- .', an. still
alivL.. S ,,li rf tl:r'-, \\Ill ui,,', !tit* ,i "i-rl-,. *,*


7---






THE CAUSE OF EUROPEAN FOUL BROOD.


sislike motion of their bodies i, easily seen. hut larva' of this same
age when sick frequently exhibit ai marked IperistakiIlike motion of
their bodies which can be easily seen with the unaided eve. Some-












IN'



FIG. 5.-Sick larva which is more trans- Fli., 'G --Iiealtily lairva' with dorsal wall
parent than a healthy larva of the [iiriir.id I-wiward ih ols.rver showing
same age. (Oriainal i lhe norr"w trantlp:lr-nr ar:ia nilong the
mdi:i dl'.r-.ial wall iOriginal ,
times the color of the larva assists in the selection of those that are
diseased. If, instead of the glistening white or bluiii,-white appear-
ance of healthy larva,. one observe, -(inie that are more transparent
fig, 5). or that possess a very
j \slight yellowisli tint, frequently
such larva' are diseased. In the
absence of the exaggerated peri-
stalsislike movement, however,
j other te-ts shouldd be applied, as
Sthe color symniptom is at times de-
_cept i %e.
Figures 4; and 7 represent older
larzae thian the preceding. These
~- have ttrned themselves in the cell
/ so as to present a dorsal portion
S"^ to the observer. The narrow and
"g^ S quite tralnsp)arent area frequently
seen along the dorsal median line
of a larva serves often a useful
FIG. 7.-Sick larva of the age represented purpose in the diagnosis of Euro-
in figure 6. (Origlnal.I
pean foul brood. In a healthy
larva (fig. 6) a pollen-colored intestinal mass is frequently plainly
visible thlroullgh this transparent dorsal area. Microscopically this
mass is easily demon't rated to be largely pollen. If. however, upon








! in-peCtion this intestinal mass appears while ,. %. hish wl-i,., the
lpre'mIIce of i1 i ropean f iIl I)rood is aIlmo-t c.ertain. A modification
,,f tli.i. -imple inspection method may oftIn II; l\ p,,c i i,, lv useed. Till-
(on.i,.t-, -i plllyv in iii'iii ii, i. ll Iarva ini the cell wAtih a pair of fr' ii ,-
untitl it, Ill median dorsal line is exposC d t the I bIserver.
M lorte fr' .iiIl.' ly still, it ill be f, ''iii I .l.iii .i_rt.eoIuIs to remove the
larval frn i the cell with I Ithe forceps. With aI little (care this can hI
done, leaving the larva intact. If Othe larva is disieased andl the dis-
ease is ii f Iicntly advaI i i',. a whitisi ilt c-ti niil ntent can I IV
often lt' a il u lnlv i-."ri\ cid. In response t I the illli-llla r act ion of the
larva thi.,- mass is ri-c'qini itK seen to e ilt, i o aI, I fro.

A OSiTIVEmVr TEST TOR TE DIS IN iDII.SE I ARV.
There is a igin represeted itn tlii.ii,. 8 which. in thie experience
of the writer. has proven thitus far to Ibe a positive .\ iii;>tni of Euro-
pean foul 1Iroi,,. When the lir, :1,,ii condition otf t lit .1,i.i:.'i larva
are favornilhe-ani1 these frequently are-the -i can be quite
ea.ilv and ilii t'1it tltI deionstraltliI in this waly: Select a larva to
Ie te.led. approximately of the ;ig, r!n ,r,-. iidtd Iy li'v tii r- 2. :,. aind 4;
rt'erio\e it frioii the cell anid place it uipoin l.i--. preferably with a
dark hIackgrtiuindI ; with a ,li>-.,tiii, needle in each hand and with
their points near (hitii,,.r. pierce with both ineedles thie wall of the
larva neari it- head, axioidini, the intes-tilne; eparate now the points
of (ihe neeilleh, so aso to tear the body wall cro-swi I-e .i, continue to
-eparate the two portions of the larva. If the larva is diseased andti
one i. .- c'..-fil in applv Yii._ the test, it \ ill be ft-iind1 that the in-
Iet..tinal content will be stripi)ld fri,, and pulled out ol' the posterior
and blind ,iiI (b, fig. 10) of the canal. bt.iiiii,, results as repre-
sented in tiguire In ca(-se of lihi. healthy larva', the inites-tinal
content camn i t he removed in this wAV.
Thi- mass thus removed from tlhet intestine, if examined micro-
-COl)picallyv. will be findil, in general. to consist of a white or -li,..'-t v
yellowi-lI-white mass ;ilii_ the longitudinal axis. This central mass
is stirroundihted hY- a substance which is more or less transparent .ind1
mniuciusilike in appearance. TIi :i|'pc-i rance of this outer porti)o,1.
however, will v:ar\- in detail. ,l.,,,lil,, in a w'...t measure upon the
stage of the disease when the exanmlitation is made.
The force which is appilird. 1 i i1,1lii thle mass frimi iph iinte-ftii',
frequently causes this "ili'iiiIIL suh-stance to stretch and the in-
closed whitish substance to break into -1,_',irii.t- as represented in It
of figure %. Thi- is ani earlier -ta.,i of the disea-e than that repre-
sented in eitlthtr or c of the samte figiIe.
38171 -C-r. 157-12- 2


'I lI -: CA I"-. .' I I 1 P',-l't \N* I I 'I L I:* ,. ,,- .








THE CAUSE OF EUROPEAN FOUL BROOD.


FIG. 8.-The intestinal content removed from larvae sick of European foul brood but n
yet dead of the disease. (Original.








If tlih ii-ci is mon re aIdv: Iii I than either -i,,it, rt r,,-,.n1 l in
figure S %% hn- this test is .I1,l1r.1. a portion of the iit1-.lirI.il content
may tl owv ,lit in the f,,ri, ,,f -.i,. the wall of which is very easily
u1 kenI. \lit 11, brkcil the content of thi( 1 alike st ru rctu I ill I1\
nut I1. a ratlli.r thin whitish or yellowish-.hite tI,,l 11 .II ,illui_- small
whi i-h ,'railil,- that vary in size. If thi li-,., is- far adv.i,. I
1niil titi larva pribli.l dead. the Inwoliii.,,i substance of the ii,'r-
tilnitl ,',iiliit is so .;i-il\ broken that I'iti.i o nly 'hitih l or yellowisih-
white 1lilid with its ,iiiinl.ir content l,,. from the ruptured wall of
the lrva;i.
Fignitre, 2 an,1 6 rirt.iro-ii I. llheathy llarvaw, anid at these :,L,- tle -.:-
imtnt., of the hly are d io A,,rly mIlarkedd ,tl' Livinl. larv:it at these
age,. if ., trelrin i, fliot, i',rolean foul I,,1 1 1 tfrvi,' iii lv show these
ninarki...- lees( distinctly as represented in li. v1- :,. 4a nd 7. Tils
S*ign, too. IlV1 as-ist inll the selec-
tion of larv'; that are -i-pcli ci l 9 U q1h U
of being tikeasd. l* *
THE V.\I IF OF' :AHl.Y SYMPTOMS IN THIE % 4~^ % 89
DIAGUNOlS[I OV EU OPEAN 1OUL BROOD. *P b l i
These yminptons of iiropeiman 4b 4 90
foul lroodi are -oine ,,f the ltore eas
import ant Ines that are id-rfiil M op 9
in sick lairvi or in tho-e onily
recently tIea d. Tlitv are c.-pt,.
cially vliimi)le in the study of v '
the di,-.ea-e in the e\|n'ii ,'lllll :l1 r t. b. llk iUhl pit.tn In a stained Vml' ar
r()lOIIV. They have not been used preparation fron -irk tirvn at Ia rp
r'--nt1d In a, fiM-r 'IT l h l
by the apiarist foir iit:kir,,_ a
diagniio-.i-. The -= ilphtnt- if' European foul brood that have been
looked for 1w th be e ktper for ilt mo-1t part are the evidlences f
dis.a-e %licih obtain as a rIeult of ii,.i death of thie ilrood. IThe
post-morter yputm- as m.iii f,-ilIv ihl dead larva' themlielves
have been the most positive evidences used by the le keeper in
diagni,,.iing the dlasea. It i, hoped, however, that when tI,,. are
well leaiirnedl. the -viIltiIII, of European f."l brool observed in
living larvia and in those very recently d,:1 may prove of value in
the apiary a,. well as in the experimental colony.
Practicallyv all the later symptoms of European foil brood have
also been ob-rved dl-initr. the course of the disease in the experi-
mental colony. Thl- fact is it-ed as evidence Il:i, thile li-,- which
was produced in tlih experinf.it:il colony was the samIe as that encoun-
tered in thie apiary. Since the dieased miateri:il fI- itikiii.L the
inoculations ha, been received fri'it various sources and the disease
produced was appareitqly the sane in every c'i-e. the concl-usion that


'*Ife i \l'-I -t 1 I l li'l.%N lilt 1 I, 100i n l).






THE CAUSE OF EUROPEAN FOUL BROOD.


tlicre is but one disease present in the condition which is being called
European foul brood is, therefore, still further confirmed.
MICROSCOPIC STUDIES OF DISEASED LARVW.

Retuinii g now to the discussion of European foul brood in the
earlier stAages, it should be einph',-ized that by a macroscopic exami-
nation alone it is not always possible to make a positive diagnosis
of the presence or absence of disease in a larva. During the very
earliest period of infection it is iipo,--ible from the gross examina-
tion alone to make a positive diaigi Such is to be expected. About the time the larva dies there is a
period at which one can not always be sure that the di-ease is present
from a macroscopic examination alone. Between these stages there
is a period in the course of the disease in the larvae during which it
is usually possible to make a diigno-i- positive from the gross exami-
nation. Since a macroscopic examination alone is not always suffi-
cient for making a positive diagnio-i-. one looks naturally to a micro-
scopic examination for assistance.
During the course of the disease in the experimental colony the
microscopic picture presented in the examination of diseased larva
changes markedly. To begin the microscopic -tidYv, it is well to
obtain the intestinal content as represented in a, figure 8. If a thin
smear is made of the white growth-mass of this content and st ined,
it is found to consist almost entirely of form- repre-ented in fig-
ure 9.
This organisii, is the one that the writer referred to in an earlier
paper as Bacill us Y." All attempts to cultivate this new species
on artificial media have thus far been ui-icce-.fiil. Since con.sider-
able information has now been obtained concerning this organism
the specific name piton" is now substituted for the Y" in the
term "Bacillus Y and the species will now be known as Baedlhis
pluton. This organism is an unusual one and the classification has
not yet been definitely determined. The generic term "Bacillus,''
therefore, may, and probably will, be changed later.
At the trige of the disease represented in a, figure 8, the majority
of the individuals of this new species in general appear in stained
preparations to be pointed at the ends (fig. 9). Some show both
ends rather sharply pointed, others show only one end so pointed,
the other end being rounded, while still others show both ends
rounded. The individuals having this general form vary much in
size. Tliev are as a rule 1 V. or less in length, the breadth being
about one-half the length. Forms in pairs frequently occur in a
smear preparation made at this stage of the disease. These paired
forms vary markedly in size and -liape. (See fig. 9.) Accompany-







1I1 1:; L .'- 11 l 'IrmA I 1 1' I.1;.., 11
i:t: 1 ,^ ,~ii i l ;' !,I,,1,,/,. K m 'lltt ;n.HIH ,',,I , i/ r I'n ,~ll,,.j|ly f,,il,,I lit thi
.,ligrv id' i lie ili'rai-,,. kut iln cmparatively small m lr,.
I f hli intent final content in i Iter i ,_, of tIhe ;i ,ase- r exam-1
i|li., tlnil represented ill I'. ti.,iii" exai iied l ieris,,,iir l ,i,' \.
fii,,'; ,,v luton is st ill f i nl in Ir lirge iiuin I TH.., 1 ',,tr iii.
SI i/li/.;' \\ hen pr' sent will le Iht li 'elV ver l 'ii i4 l('tris III in I Iiii -
l1 ,'-. .\ -inilar exa nti nat ion 4f thie ilt(,-tin;il (ftt'iat rfi'resfntid in
fi. ti ,ire ,. will IIIUI lly sh








0. -


/'. i ', .ii i ,,,n l l "y 2 t n <





Litl
1 "1












I'*a';. 1'i .",. l. ma~lk dra I- TnI rlr',s'n i Inii i n ,, r t an TIrl -
t.i-'i. ,f I 1'M1't oi The i n-,ir ibi l,'rltrophlic nimbran., (Originaln i

By exariiiiiiiii th ttlh liias which tl, 'v- fr om the body ,i f a
la1r1i \vlw,, the disease is far sadvt;, I .l ani M i le 1 vdy wall is ,ii 1 k l ,i.
p uie usuiiilly fili t-,a .tli.r w ith i',''/li'.s id tr,. bacteria of ditirliriit
s.peci.- in considerable nuzmlnrs.
Froim tllIs plixit on in the 1rcivaY of the larva the relhilive propor-
tion of the different tit rDir.II-In= )- present v\:; i- markedly. WIiin
Bawll,,.< ,1,'; is present it increase very ripilly in proportion to
thle olther-. TIlli r;,lii increase of h',, //'. alw in til- larva a ifcr
thle dea;il t' the larva* accounts in a large measure for the freiiiienvy
with which this species is mentioned in reports on thii di-,c,:.






THE CAUSE OF EUROPEAN FOUL BROOD.


In living larva,. therefore, in which European foul brood can be
diagno-ed from gross ex:amnination. it is found that bNcteria usually
accompany B,;ll.its, plutom. This fact made desirable thle study of
the diseased larva, in still earlier stages of the infection, i. e.. during
the period of incubation. This was done culturally in part. but prin-
cipally by fixing and wectionin-g the younger larvae froinm -trongly
infected experimental colonies. From such sections it was observed
that Bacillus pluton was the fir-t invader of the healthy larva,'.
Figure 10 represents .chelatically lthe condition in the larvae at
an earlv ,t:it, of infection. In this figure ',/ represent, the foregut;
,n, the minigiit. and hy/ the hindgut. At this age of the larvae the
posterior end of the midgut is closed. a- represented at h. In the
same figure, m represents that portion of tlihe intestinal content lying
in contact with the wall of the intetine; : f. the central portion of the
drawing, represents the food taken at this age; and p represents
what seei,- to be a peritrophic nmeImbl)rane between the enveloping
substance, m, and the paplike food substance. J, of the midgut.
In the growth of Baeillus plenon this parasite very early takes a
position along the peritrophic membrane p. and just central to it
(fig. 10). At this early stage of its growth this microorganism pre-
sents in general an appearance of being rod shaped with a strong
tendency to groiw in chains. As the disease advances and the growth-
mass of this organism, increases, the central portion of the lumen of
the intestine becomes filled by a solid growth which is made up very
largely of Bacillus pluton. During thi. stage of the disease the con-
tent can be removed from the posterior blind end of the midgut, as
shown in figure 8. The relation of the central growth-mass to the
surrounding mucuslike-appearing mass represented in a. b, and c
of figure 8 is well demonstrated microscopically Iby sectioning these
intestinal masses.
From the studies made thus far it would seem that Baiill,,q pluton
is easily killed by heat.

PROBABLE EXPLANATIONS OF ERRORS AS TO THE EXCITING CAUSE OF
EUROPEAN FOUL BROOD.

It is quite probable that others at different times have ohberved this
new specie:.. B,, lu., pluton, but have failed to differentiate it from
bacteria which were present and which appeared in the cultures
inade. leading them thus to erroneous -tatements concerning the dis-
ease and its exciting cause. For example. William R. Howard may
have seen this organism microscopically in his so-called "black
brood." but failed to differentiate it from some bacterium-Bacillhs
milii or BR ;lhi.. alvei-which he cultivated on artificial media.
Burri may have seen it in the so-called "sour brood and mistaken it








furt.1n- O WL Mfiti -11",i- "hwir I e I b 1 ved"Inn IbI is cltures.n Nllndl-- 1,

*,, IIf /flM from lii 144 w hich on mii~rU-scopic( ex 1 itiittloio selemed to
contiitni i f this ba\t\rium. Io r i 'I:iii t i. I I. iii ie advaI t li( I 11
sniipo1 -itintr tl:iat the Si I t''ili' ii', was ri; 1.,l Lill.I hv .1, 1.1 pro
dic' I lIv itself. T1'hi dinir.il'\ promhly ,uhl IIt as 1w l ., R .I ,ii N ,
bIv -, i, i,,,.." that .X i::--,.i t'.,,lId to d ilo i tiliIt .tviur iumll whlifh lihe (rltivitold maltd uh y'rilkd !
IS 'I III-.l+, MO(KIK THIAN (>> IH A IX\ 11- g\, I\ TI1] (*I V IC THIN IM 1N \I N AS
Yl+ RI % FtU I' I ltV ilmml?

Tih' (Ili]' ll-( now ari-I(. \lvitII-1 or I- not thl'wr is lionr thai olie
,iii-..-, in the ,1111li, il io \ now klnowlin :I F' lil'. ti ii folul lrwoiod. In
S .it/ i'hliii aind in ( 'lriliii i tiheri lils |ici l al tenIid4 1ncI y tiJ 11:1 iii,-r
tilt di-'t -ciI broodl in wliich ]: *./1 <,il j is i',41 ,,1 us th1 foul ir.,,iil
of ('i-li- irt. anid ('huivii1 and thel dis'aased brood in which /'epfto-
1'11 11,, i f ini, is sour alrood." "r i ti ait fi- at ha d the t II t
ivrilt er is strin'lgl in.lidii l to believe that thte- tw\o condlitions are
,il" y th one ,i --i. known in Anericai ais Europea in foul broilod.
Enoigih evidence hais not yet bee"i M1iAW iiil. however, to pvakl with
CiM As .em 'idiiary invaders some (if ilhc species of bacteria mentiiioned
in thli, p ,per lillay aildI probably do exert an iiIIi,.'i.,< on the m cours
of tihe disease in the larva and in the colony. To what extent these
alitieriii imidiify the disease is yet to be deteriiiii.lI. Sli,,,iiI' it be
fund tlhit B-, i/,.l, il',ei actually cauiseso aln ilnfectiois brood di-.-v:i ..
lien siieh a disease should be called I-'.iiropean fAiNl 1h,,,nd. and tlie
di-ease enwilii-d bW Bfl-Wn pluton would have to 1be dithr',i1ii,.,1
from it.
Fiirthelirl detail. will not be _,ivi i in this 1)reliminairy ainnonlce-
iiment -lit will be included i liol'e technical paper which aret .'-
plr'e pil rd.
S l IMARY AN)D CONCIINlNS.

The -01i'- taken in thle writer's endteavor to find thle caiiusie of 'llro-
peoin foul brood mIa;i* be 1) ril slutninarized as f,,li, -:
(I) I i/,,,''/ .t+ 1?'. ,, which has- been so 'i'uiir,'lly spoken of asl the
cilli-, if fiIl !'rnl l. was i-ilhild friii li-'.i-'l bIroo l. alld pure cIl-
liirt- .f the ,-._,r;:ivi-ini in bWth the Vr ;I, t Iti V, ani 1 poire formIs- were
rpea litIdll ftedI to colonie- of healthy bees with the result that foli
Il-ronod was nIot lr,, ,lii'd i a in n liv instance. This fta, I cast a s-s-piCioll
thatR /'". ,/,. t/i was probably not the cau-e 'f a di-sease.
12) By a study of many larva in -..ori- f otl:'iropean foul brood
it \via- fr,'lii-,iitly found that there were a1irv apparently dead ',f the
Tli.'-a-v thia cointaineLd Boi, '1/,' al.e; ul]v in small nulnbers or not at


1["l. CAI -I OF 11 I;,,1'! \0 014 % 1 I L l loiil .






THE causeS E OF EUROPEAN FOUL' BROOD.


all. Thi., increa-ed the suspicion that Btillsix ,/'e; was not the
exciting cause of the disorder.
(3) In 11107 the writer proved that by feeding pure cultures of
B,, i'l,,f larva to healthy bee, American foul brood could be produced.
This fact still further eniphl:i.-izdl the doubt that was already enter-
tained ',nverinieig the )possibilit ie- of Bi'illim.s al,'i( in the etiology of
European foul brood.
(4) By feeding diseased lurva- to healthy colonies it was found that
European foul brood could be artificially )produced. -.,howing that thi-
di-ease. too, could be produed hY feeding. and that the virus was
contained in the diseased brood.
(5) The sick larvat of the dieea-e thu- artificially produced were
frequently found, when examined, to b)e free from Btrilus al,,e;.
This ex'idle'e, too, was dam ,aginig to the theory that Bu ;llhis al'ei is
the cause of a brood disease.
(6) Bwacillus alvei in this way was, tentatively eliminated from tlhe
list of possible exciting causes of European foul brood. In a quite
similar manner the other bicteria-p.Sferctoe,,d.,. ,/ai.s. Bua-tllus mne-
sentericus uiilqa '., Bacillws orphleits, a(Id Bacteriim in crydice-were
likewise eliminated from the list.
(7) Considerable quantiti.- of tiltrate from aqueou, suspensionss of
crushed diseased larve were fed to healthy colonies, and in no instance
was European foul brood produced. This eliminated tentatively the
probability of there being an uiltramicro-copic virus in European foul
brood caplible of producing the disease.
(8) Bacillus pluton, therefore, was thle only factor that was not
so eliminated from the list if pos.;ible exciting caties of the disease
and became thus the probable exciting cau-e of European foul brood.
(9) When this organism was -t died in la'rvse in which thle di-ease
could be suspected by inspection alone, one or oI ire -pecies of bacteria
were sometimes found to be present al-'o. The-e, when present, how-
ever, occurred in relatively small number-.
(10) The disease was then studied in a -till earlier stage; i. e.,
before its presence could be detected by gross examination of the
larvae. This was done by cultures in part. hut principally by fixing
and sectionina larvae bringg the inciubhation 'period of the disease.
This study demonstrated that in thlie product ion of the disease Baf4lis.
pluton was the first invader of the healthy larve.
It will be noticed, therefore, that in the determination of the pri-
mary exciting cause of European foul brood two objects were accom-
plished: (1) All the factors in the lit of p)o-:ible exciting causes of
the disease were eliminated except the one organi-m RBallu.q plifton,
and (2) by the study of infected larvaw soon after the infection took
place, this parasite was found to be thle first invader.









As it ,oi,'ll-i,,ii. it is iI, hel Wief of the writr that -,illi, iii evidt mi '1
lIii. ,, i,.,,n-n ,,1:it,'ld to justify the taUltimct. thai 1.',,* I/,..* thl,,t,
i-a, l,. ])riii:, \ ,-iiiii._ cIiu -Ie of I irt l ,li-,.,-,. "11,n- Ii ... di)-Ise

i. lIfi, L!I*_I,*I,: llyv kiii,\inwi ni Auncriiai 11 liiroiwaun 1'mil irood. Thin
, i i,,111 i~- ,iihlrr'1 iII act ornii'. e 1 ith views now 1.,ll \lk'lly ac\eptMd

Al.l I I l iil, t ,f I iii-itiit Il I t
"'l1'Ii ',' .,ir,. lil in tlirwt lirilti l,,i* 1 ~l' ~ ll-,i~si-,s. T w o of thlts!, -
.\ l ill '!ri( i I I li n~ l I i ', ,,,'l t'N li^ i'tl l V / .r i f f .f < ffff V ii i d i' ii ri l U.oiH i n I ,, l
',l1,,Ii i.i.ii-.lil I'. ha,, /iftfs p1lIut -ii- iIrc- kiio l t< I ft t iit iiol. "Iroll
ii ,-t' t\i > i-rses iliee' inlll4 I *Ii il i',. nt ii t l ihe third ',it,. ian
iIipir.ilIV iii \ iiinfectiiil i ri +r. the s< ,il,'.I "I pickled lwriod."
I.iir'';, ,I,.:II iif this latter di'tarit> :lnc |ri , L 'T illii- lii "1 li ,'\ i li li ,L '" in i !e o f l iis dli-f i'd ht r i > iiotl V f t k lio w l .


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UNIVERSITY OF FLORIDA
IIIIM 12210921 5t7ll9311INIII l|
3 1262 09216 5793

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