Citation
Eimeria tenella: comparative pathology and lesions of experimental infections in bacteria-free specific pathogen-free and conventional chickens

Material Information

Title:
Eimeria tenella: comparative pathology and lesions of experimental infections in bacteria-free specific pathogen-free and conventional chickens
Creator:
Radhakrishnan, Chittur Venkitasubhan, 1937- ( Dissertant )
Bradley, Richard E. ( Thesis advisor )
Edds, George T. ( Reviewer )
Hoffmann, Edward M. ( Reviewer )
Loggins, Phillip E. ( Reviewer )
Smart, Grover C. ( Reviewer )
Zam, Stephen G. ( Reviewer )
Reynolds, John E. ( Degree grantor )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1971
Language:
English
Physical Description:
145 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Bacteria ( jstor )
Chickens ( jstor )
Coccidiosis ( jstor )
Flora ( jstor )
Germ free state ( jstor )
Infections ( jstor )
Lesions ( jstor )
Mycology ( jstor )
Oocysts ( jstor )
Species ( jstor )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Poultry -- Diseases -- Research ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
In order to determine the role, if any, of the indigenous cecal microflora of chickens in influencing the development of and disease due to Eimeria tenella, the pathology and lesions following experimental inoculation with a standard dose of E. tenella infective oocysts in bacteria-, fungi-, pleuropneumonia-like organisms-free (PPLO-free), specific pathogen-free (SPP) and conventional chickens were studied. The dominant microflora of apparently healthy conventional chickens and changes in the indigenous flora following infection with E. tenella were studied in chickens aged 1, 7, 14, 21, 28, and 35 days, using standard microbiological procedures. Experimental exposure was carried out by oral inoculation with 100,000 surface-sterilized E. tenella oocysts alone or combined with single or multiple species of bacteria and/or fungi. No clinical symptoms, mortality or gross lesions were observed in a total of 32 bacteria-, fungi- and PPLOfree chickens inoculated with E. tenella alone. In these hosts a retardation of the development of the endogenous stages of E. tenella was evident. The presence of a pure strain of Bacteroides sp. , Clostridium perfringens. Escherichia coli. Lactobacillus sp. or Streptococcus fecal is or single species of the fungi Candida albicans or Mucor sp. resulted in mild cecal coccidiosis following inoculation vjith E. tenella . The typical cecal coccidiosis syndrome developed in chickens harboring 2 or more species of microflora, viz. , C. perfringens and S. fecalis ; E. coli and S. fecalis ; Bacteroides sp. , C. perfringens . E. coli and S. fecalis . In SPF chickens, typical cecal coccidiosis developed following experimental infection, with a mortality rate of 3Q% and a mean gross lesion score of 2.6. In conventional chickens, the mortality rate was 22.1% and the mean gross lesion score was 3-1. C. perfringens was isolated more frequently from noninfected SPP chickens than from noninfected conventional chickens. A stimulation of growth of C. perfringens and coliforms occurred with a concomitant reduction in the grovrth of Lactobacillus sp. in SPP and conventional chickens suffering from typical cecal coccidiosis. The results indicate that certain species of the indigenous microflora of the ceca are essential to produce the typical cecal coccidiosis syndrome, following ingestion of E. tenella infective oocysts.
Thesis:
Thesis (Ph. D.)--University of Florida, 1971.
Bibliography:
Includes bibliographical references (leaves 124-143).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Chittur Venkitasubhan Radhakrishnan.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
029884477 ( AlephBibNum )
37704197 ( OCLC )
ACF3896 ( NOTIS )

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IITI;Eih: TI 'I,L'.: L"iiV PAJil.TI',E FATHO!'..'G"Y AUID
LL: i': li'S OF E-'FtI.R IlEii'TAL. iJREC'i'TI IS l
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A Diss. t' tL i'-,i Ptr.S-: n.-I .'l t h' e Glji..,l.-,te C ourj i.:.l of
T:he U',v- 1 L .' :. I : Fl: 'L. In Fai t ijal iPu f illcriint of
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A I: ;: L.EDGi [C!ITS


'Phic tholr e::prTe-::: hi; ?ppre.i-i-tion ind deep Zc-nle

of grat-iPtd tro Di Richr rd E. Byad :,, Sr., chr-iirian or

his : .i ,up-rv izcry Co mitttee, Cor l: I pful ii ,g!: .tio'l, c'oujis.el,

ad3i enco uriI '3 :.C t tnrlrci hiioi t t i i 7 lii. a 3ri d ring preprta-

tion :iof thic di'. rrt.atio H.:e i oc. irid:-bted to Drz.

I-. T. L .i:, E. i. Hoffiri' n G3. C. Sruart, Jr., S. G. lam,

anid Hr. P. E. Loc'vin:, r:'iber'- of i1n SuoerviLory ot Committee,

for advice. e ard -Uid ce. I.eci :i tn: n' -: for help i-i r,-in,

Ja:.'. io tI: i;r:. W,'h PFre;t.s e i .1 a i r-iloi .-adut1- e .t1

rir. David J. 'Jei r. [I'. Louis I!. Crtle -ain [Ir. Jame: E.

Dic!'ini-s on, Jr., proi- idel teclL iie.ic l help for iliicn the

aut. ior its iindebred .to tr. m. 'r3at:--'ful th'ankc 2 ,c to i. H 2:

.Jean E- irr 'rfy r fp:-cial t-c.uiic:l I l.p. T'''ioo fa' ulty and

'iP-adli.uate stiurint: in thl- D:p--.trc: nt of' V'.t ri3tar" 'S ien',ce

ha-e a3.so b.--:n hcipfui i ,. ,' 3 :.77.

T"hi .autth .r t;. i .;i :- o a .* . tu i- -:t:':o sd-o.:. ti :

finaricial i nppFort 'of 1I D':. part 'i it :1 V'aetitriy- Sc icienc

in p'ro.idii:" cra3.lat' - -,-- 'l !lp i; suip-

por't d thir throuhoi: ti: :tud, I'l, r :r .--. 1 : c7rrie

ouLt : a '1 prr of ,- q*ajh ;- p o... ci ..-e C '.-10 O ),

ent titled "Biolog cIal:. [ietnid or i '.,ntroi for I 'a li









Pirasi i i:es of Livertoc:l:. He i:- greatly indebted to his

'.;ier, Jaya, for her help, encouragement, and understanding

throughout the entire cour.'. of thick work. Thlanl: :Also

ar extended to [lrs. Jo.vce' Jarvis for typing tie manuscript.

















L I I'S L D,1 1 I'S ......... T........ . ................ '

Li i '; .,F TA E. : . . . . . . . . . . . . . . . . .'

LI 'T OF F I-jRiLES ....................................

. 'i i . . . . . . . . . . . . . . . . . . . . .

Ii fl 'D ..i;r . . . . . . . .. . . . . . . . . . . 1

LITE1L T1.1T E iiCE I fI ..... ............................

Ei- i ri rr -, : L i -.':l- : i l,.'orp o .....

Sh:: : :f E. r -i Ini ction .......... .

P I .:' ..:.f '.., 1 C 1:. l 1 .... ..... .. ... 2 1

_crf ,,.lo -ri H, t .. ........... ... .. -
i G.etr-Fre C, ic: ....... ......... ...... 1


fP Licir; .T' IAt' .- inril F recite: and
F. .: i i i .- i . i :f : : t -t . . .. .. . .. . .. 'M

i'R[l'L Ii .CROF- I L F L.,F ." F C'HIC L.'. .......... ...... 0

I TLERI,'L3 ;' r ;P'iri'.,D ........................... 44
Sl .. '" ..! .. . 44
i. ,,] t, on ,..l nt 'jl o'.. C_,,.r .4....... 4

II. r i ion of i P L l: L-- ....... ........ 'i

III. F ,ri.Jo ct.' .r, : t j ..] 1 'iick '-t n; ...... .

IH. 3.uri' .r ..-- 11-.. ....... ... 47

V. F h l ':, .;l . : .... .... ... 4

V I. : .l- i ................ 4]

A t. h e,:7 r i -. i j ] ,- f -
rth e c" u,-,', in ,. *-*:n t :l .:. ,i : li'.. i' '1 . . 4-








B. D.-i.i.- in-t l fi n of nic.riobial floor 'of
thi. c ucr-,i Un -Ic i-f'ree SPF, and
co I'.'.: it I cl. cll C :e,'-. ... ....... .. 51

VII. P-.c cd'. Ccll 'olume ............ ........... 51

VIII. Total Sc-rum Protein ...................... 51

IX Sr ru.i lec tr ph i ................... 53

E :posur;- to Ba' terma and Funl;]i ....... ... 54

RES ULTS ......................... .. ............. 5

D,.cve oop -.nt or 1licrobi. l Flora in the Cecu'n of
Co v:,', int io' -,i l' 1 ,icL:!- Inoculat-d i ili C. t rc.- 11 a
and Uil inroculatc.dl Con Itrol. ................... .... 55

BA.t.-ri I Isolates in 3-'ic l:-C-1-I SPF -,nd Con-
Sc 1 Clii lc.i In'cilatcd t.i th E. t.7n r.i
cntio ,l l Ci hii et :i C c.c lt'n o d ith t... : .

I C'J U.IoIl.t. . . . ................ ... 6. .




DI CU PFL:: DI : .................. ............ ..... ... . .
S'.JR:u Ft Y '.,D CPCLU' IO3 ]S ............................ ,9

A PFL i: DIX A ........................... ..............

APP2i DIX' B ................................... 12'j

DI LIC 'G:. P IHY .. .... . . . ........... i..... i

EIC'. \PHI.;.A L Si'K TCH . . . . . . . . . . . . . . ... L:














LIST OF TABLES


'Ta.ule 'sPce

I Fr ,in r;, I ,l':-it ion r.:din iU.e7 d ifc.r the
Be:c:'v r' and Id:-n ific-.tion of ab'ctcris
n-id Fi.mi._i frm C:-c ofr SPF and Co:n-
*.;-int ii n Chicli:iel.n ..................... 52

II Cecal Flora of Y':c'.nr ?c'.et ional
C hi.':,:.:l E:.:too.-, t.a : i -er, il ten lla ..... .I.r

III Pr-c .orl'i nant eacte 1' 1 "lndj Fua.ral 3rp..cier-.
fr r Cnhic Ie .3 F .n c h i'inF Lc c-.c!l ,s f' Cc'C l
Ccccil :.i 2 icr1 t'r:'ri i'oiexpc.sdc C n-
vciz. ic n l 1 Chicl :ens ...................... 4

IV Fat~hlC, o,i,' DI.1- to Eiii.r'i tm-nell, in
act :lr Ia-, FtIirl- :iLd l' i.- pl i,.u oni -
Li\', Or.c'ni-,ii p -Fr.: -c Chic'.1r ns i ai.'-ed in
Plastic Film Ic1-.l tor: .................. .0

V F?.t:i ilo- .' Due toi E-, r i t':ill lia -:Ind a
in,-le Sp cC. I- -C f Finr i.i- 1n t-._acteri -
Fr'e Chiicl:.:is a3is.ed in Pl: I tiC Fili
Is c l t r. :, .................. .......... ... 5

'VI tf'; i'- loi ', Due t, Linr,-lr ta r. :lls ai' a
,Si le Species n'-f c teri : in Chi.celns
fi'-. P.i in Pla'tic Film Isclators ........ 0

VII Pa- thol.ilcr. Di.i .e- tO. ir.F- i- fill in
Chi,.:en[ R'i:e. in fla r -.c Film
Icc _l: r- P' -lyc': 3r-i ta 1nin t..-- u it
.c ri an 31 : r Fi. . . . . . . . . 4

VIII P-'?.h .lo '/, D t.:- 'o Ei,,r-ir i t.r, -!1 in
Ii :: or 'i-Fr' :- C ~c ......... 1


C'I.r lnt icoiil C ItC!. c... ... ................ ... 1'

occ, r trperc c. l C1 ] t 'i. .i 1 , . ri,.'r-', . 1

F r: :i.:i ':., rL*'.-. i :n:-11. C iici :' !. r. r,,' ;.ed
to EC r t 1] . . . . . . . . . ..... 110








Table rsa.{

XI llean PacLed C-11 V'ol-.e (PC'/) of
E.F ct ri- -, FLn 1-'i-, air-id PF'LO-Frce,
Spi-':: ifc PatLhogs-n-Fr-cc (SPPF arnd
Ccjnve; icon l Cf ii:kllcrn E::rofed to
i 1t f '.- ] ....................... 112

XII T'otnl Serum Frotcrins -in- Seruri Protrein
Fra~ctinc in Infct:.d and Hionitifected
Ci :,'crentionlal, Srec- if iO Path 1:W'-.n-Free
(CPF) and BE.ctr-iia-, Frunci- andi PPLO-
Free Chicl:en ....................... .. 11


*. 1 1 L


















LI;T i:.*i, fli,].li








f .- -,7 . ...-- r. : .-I' .. I --
il "+ + + I. . l, . r. : .' . i -' . .

I, -,- ,, r ...1 .- ,, r , : ;i. .:. .



H+ T . . . . . . .. . . . . . . 1
l, I _- L", ;," : tl7 i r t r,,-'I ] .i- ,_,1, I










S. . . . . . .
..i... . .. -I .
in +++. ........ .. ............ ......... 1-
I- - I
-_..__-: ._. 1_









,__ ,- _____ _. -


.: -,,_ ,. , .' ,- ". .. ',.'
.. - - ,



I '
I j I ~ I- L' -, : 1 r, 1












I I
+ ]'--'L. ... ..... .... .... ... ...... .. 1







A atr act of Dizsert -ti on Pre-ent,'d to the Graduate Council
of the Universit' of Florida in Partial Fulfillment
1o the REquiremints for trt:- Degree of Doctor of Fhilosophy

EI IRIA TF" r:LA: CO;!PARTI'/E FATHOLOGi AIID
L~ IO: CP E:-.ZRIY:.:T,.AL liFECTIO!J3 II!
B.\CrFr;IA-Fri-, STLCIFIC PA.-THOGl-
FREE AI!D COINVElU'IOiJAL CHICKEil3

Bv,

Chiltur Venl'itasubhan Radhakrishnan

Dc-emober, 1'i71

C}-iirman: Richard E. Er:.ily, Sr.
MIajor Department: Animal Science

In order to determine the role, if any,, of the

irn.lijgenc-uc cecal microfl-ora of chickens in influencing the

development of and disease due to Eim-r-ia ten'-lla, the

patholo.-;y and leionsn fo.llacuin, exi':rimrntal inoculatirnn

uith a standard dose of E. tenella infective oc.c.c-ts i.

b.actoria-, fungi-, plut.lropneumolnia-li:e organiirss-free

(PPLO-free), specific .athogcn-free (SPF) and conventional

chickens were tudied.

The dominant nicroflcra of apparently. healthy con-

ventional chickens a ,nd changes in the indigenourZ flora

following infection uith E. tenell were studied in chickens

aged 1, 7, 1t, "1, 2., arnd da:.':, u.-inc standard micro-

biological procedures. :peri:,-entLl expor.re carried

out by oral inocult icrn ith 100,000 surface-ct cr1ilized _.

teneila oocvyt:t ?lonic or cou;.if.ncid 'itn .in-le cor multiple

ope.:-c of tbLctcr>-, -r'.'-r fun -i.

1il clinic.:il symptom:, micrtt.allity or gross lesions








n .:rer ,,:.i i~ i rictal of 2' b1r.~it ri fur..Li rriL FFLO-

i'''.e c. l 'C.L i: irinc.: il-.ted i: h :lrL-. ten-ril 1 l,:'I 1 i'I: i. I ihe.e

rn ost- ret fiardat Io.n :.f trhs d'v:. lopieritt of t, endoceqlI

st.ajz. of -. t.-i:' :ii: evi.?ert. Th.: r:. n c'.: of a pure

:tvr-i o E'-'I- P.'IL'-'r -. C, l '-t' r i, r.,-, r-f ri' .en ,

L..:l i'iIi -i .c011i, L-~ ,: i:t : 'illi? p. Or 1 tr [ rl'-,:l ci:: ll

:.1 or sinLr -l '- 7'CC C Oi f tih fru-. i C i' -i i l1

o:r i.:or i .p. resul:te-. in mild 'e'al c:icc diosic: 'llo.ini

iitc1l, :At ion fit E. tene]la. The typical 0 :31l Coci.io-

.is E.'yr:iiori.:s dev'.'lop.d in iic':>.T : .l:'..orinr 2 or nor-

z Fe'ie. 1 of imicrofior',v ., C. r-'rfr i .,i: arid S. fr.ca i0;

. c.- i and S. Cfe,-ii ; E.,-ter i:.-le .p. C. r,1rfm ri'-ns,

E. colt ar-i 'w 'c Ol is. In CFF :hicl:-..-n typical cecal

co.c'idi,:.Eiz .-livElop .i foll,.ini :.:pe-? ,1r r- t.11 :i -f.c ion,

jith a mortalit: r ati of 3.O and 3 -.'n :m.oss lem C, ore

of ... In conventio:ar. l *Zhici':n., t m:.'rt:llity r,-ite w'--

' id :j th mejai ,: i ao.s lSiic, score? i.as ,i.1. C. -

-'_i_'__ ,,-__ : ic.:1l .1 t- I m, orn'c fr aie, ntly front, n.:.ni!f ct,-:.- CSPF

c ic.i:.:c i tiisri fr'.r) noi ninll-'cct.ed c.fln,'-ntiontals chickr,-:. A

t- imulat ion of LL'u'.th of C. .-, fr'i r"-' and coliform;

oc .l'rr d lt'ith a ,'o L' i t r*. : ri'i in tah"- o'I routi of

L-,rt.. i l us sr. in SFF rnd .:*Wn,'Ert .n-il c:-h l::cin CIiOf F-

inrt, i. ,' ypi'?.l C:c:-il c.0c:,. 1 :,: .-.

Th- r-.su] I.2 iniiiat,: th'"it ,r-rt'Lirn ;p cies of thL

ilio ii .?i' icr :i ': "r-I :t',. ':t-. i. al to rT.ojdu

the t'"pic l c:a-' ]. c,:ci.c.i .Lo: :".tdro,,:, follo'iith, in ::- rio.

ol F. tr'-:n.:11i irif ctiv., ,cc., ::tz .













lilTRODUC IC, II


Li .- ria tf 'i-ll [2Cillot and Luc-t, 1891; Prc.tocon:

Eiracrilda-] is the mort ccn-ion and pathogenic of the 9

species of Eim-i in described from the chicken (Callus

dom-., tit). All the 9 rspc ies of Eimeri. occurring in

the chicken ar- initrc:.:llular parasites of thc epithelial

cells of their intcstinai tra.-ict producing the disease I:nowin

as ccccidiociZ.

Coccidio-,i. is a dise.ae of great economic importance

to the poultry industry tlhrouahcout the world. In the Un'

State alone, a total lo:z tu the poultry industry of

.3,;j:',000 was -timaL .. during the period 1951 to 1960

by the United St-it.ec D-p.artment of A.riculture (1965). Of

this cum, :015,123,3000 niere attributed to mortality and

,8'.19,7 1,000 to rnorbidlity. In 1966, the c::pcr iiture for

coCcidiost0-tic- ICu,7s in the United States ais estimated to

Db tetuu',11n '.'~0,00), 00- 50,00 ',0 00, for broiler and laying

f'lo-k r.:p.i c lene t rhic'',ns. ,. LuteL cocc dio- i.: nith a high

rate of inortalit" is rc ul1-1:; a-s,:ciatc I ith infection

due to [. L.1 i- l and cin,: e t:'. 1-1.; i-.:-.- -.- .onfined to

the c.:ci. th- dizc'?:"Le is oft' n ref5. red to as- acute -ecal

o- :' i..ti . Yo :. ch ic .:, : 3-: ,',-r.': cf nig.: Ii'e highly

suaceptiblI to t. his di'ea-.e i.Lh 1 real susceptibilityy at








arbiLiL ':.'-.t I (,:'if 'i:' it'1 l'fi. ': I'5), 2 r v=r :l c, trer

.auiit1hjrs 5i s zuie- "t t 'hat V'or,.i c1 ic I:';i ,: ic or': su. -

c eptible than older :rne (Ty:-'er, 1929-; K.rrann nrid Prc'li,

1.'33i Ccomn.ermely, mar;, ctn er i: :ii-,ect that th, r.v- rse

is tr.i, (T z::er et -l., l ?- i;:,-i u;, 193'T; .Jcrnis, 193;2,

Hort:c.in-Sithi, 1' ). It is impFortant to diiticicuisl between

cusCeptiobilit:' C.' hiC:kenl t:o linUcal dien:'-e anid 1u.-

c Eptibilit- toC *:, a idial iif'ccr ion a, .n:: fl;il*'d ti.:: oc.:,g;t

prc.du' ti :i Based on, c .:; t Frpi; duc: f ti,:n, clder' Li rdis are

n:.r:- usce p? ible to the p'traza.t thil L::: c.un*er o.r-ne

(R sc 1 .":'l. T!i 1 is dlii t:c the hi her rate :f ::; :,c t.i-

tion Cof ti. c ocy s : ; D':Z r -In andFI P rr, 1965; :*e, l'c 7r

Older bild? are -ilsc su-:iF. title t: lini'al i'irpecticn built

tli,. rate- of mcrtalit, is isuijlly louI diue to rin :pprarerit

;-uiioipr'd irimunirtv ftr pr r vic is c.r-t ..l *..,posures.

Leviine (,1r6 soe.losJc ted tiat tne acquired immtiuit' is often

no.t bzoliute,, bu..t :enerall'. oi', a c: ni r ition f' r elative

inimt.iit',. C'c-:-dav,-oJl r -u:ce -ol. .i.*ckens .'are le s

i..: .t i ble to t ir i : i i.'::t 'i2i r B in *2crr p irl..L to c[hi.c. ens

'-5 'I+dl:S .i' 3 age ( :.e, 19? 7t). Tlu.i thle severit t orf Ilhe

inri'e'ti :..d. dI:w c:.- in cv'i:::c:n i'.lcr f,'.Ild :cnliLionc

is r-:l. -1 t to ,'-e an~ j brL:i.l 'of tli ch11'?k n1c rr-evi:'iosI ex'-

rpo nrc .fi tl .--r'c-: of :.p -' i.'- t.:.. ti infective stagc

: r tlr- p r:.:ite. ,.t the pec nt tice, co.: :li: i i1:

',i, .1l l i .; t.i riO n: ine UiC.e .C rt .i t:o:c idi'e tl i.

dr, Ii. -',: pr'act i -' l _Fo.r *.;'-': ni.: rc-, ,: tlese iru-:-' ha'Ve








to be incorporit- d in the feed or dzinkin-c water of the

chicken from the da:' of hatchinif :nd continued throughout

the life of the bird. This continued use of diiruic h1as

resulted in intcrfei'ence 'iith immunity (Davies 3nd Krndall,

19 ; Reid, 1"160), side c.ffccts. su-h as reduced fertility

(Jolmer, 19m I nd development of drug-res 1 ctant strains

([!cLoin.iilin and Gardiner, 1961a, 196'.1b, 1 62; Pcllzrdy,

19ol 19c22, 1962b; Gardiner .and IlcLoughllin, 1963; Vegh,

1963; Jo'n-r, 1970; IicLoughlin, 1970i. moreoverr, drugs

preccntl.y available do not offer effective protection

a iinct all tha species of Ci-v-ri- pnrasitic in chickens

sni nmost of the current brond spectrum coccidiostats are

not suitable for prolorned pEriodr of use in chick.-'n

intended for ihuri;- con-.umption. In spite of tn,' hiTh

efficacy of modern coccid.io3st.tE, outbreakl? of the disease

may occur (Joyner, 19',0) due to highly levels of contami-

nation in the environment, reduced upt!.:e of the drug or

development of drug-resistance, and a high degree of suc-

ceptibility (Joyner, 190, 10 71.

lUmil-ri natural c-ndi tions, cec:i] coccidiosis occurs

thiou,hl in,."r ticii of 1 -irge ni bers a,' th.e ife: ive stige

of tLr-C:e .l called spo;ulatcl ooc '- r Ti. unnl:.pori'l tcd

oc.c'.-:ts uhi'.n re forJ. I ed in t': ,nitheli-.i1 .lls of the

ceca are c:!pelled with the feces. The: develop and sporu-

latc ni the ;-rou: ,l if con:liti.. ..' '.-f o:-:.';:n te.'t ion, noise ture,

terip-rc-ture, a.nd other environe-intal factors are suitable.








Th.; si.or uilite. c.7oc'',t ?oint jitu i~ :. zri ro:cir.- 'i tlhe

infr c' ti .ve : t -,- The fir.s ste. in t -he raLiiO. *-..:- i o

th,? di-7 ':rei' iF excy:t t tion of tih' i;i :.ted .oc t- Z..e

Ii'"'.r. f'otluid rapid e.:c r.tat icn of the naijoritl.' of tlic

C..:c: tz in hii hl,' ?u. z,'epribi caiii n: 4ii; 5, oi .- :

oid, whi le:- :u:cc -z ful p::,?, t t.ioni a .d lou o :-'St pro-



le0.: :u:c ptibie to ,ecal co.?i.dic i- Sh.: :.?ritc the

r?,: for vevy iou prc.ientae f-" ::,nstc:.t:lon to in-

m 1t.rit. cf the ho, t: ( i eas. action .c. tle _;zzar.1. *'all

,Iud cutb- :pt in l conil-intra ion cf tryptic j,.i:-;. T)l,,r.:

are -i 2conbination of r .-ctor-, tiec. ?ar:' for E :.:. tsat ioC

.:'ch .:-z bile and rpincreatic i[?? : (Lxvinre, 1 -'2'; Ik!eda,

195.;o Hibb-er't et f : ., 1nQ).

Our imr..:,ldger re A:.r irng the role of iite-tinal h 'l _.

in the initiation, E:veloprient, or ev.rit:.' of .:eCal ,oci-

iosi is limit.1. Ho.;rev.:r, it ha; been r'?.or t;d t:'y

Joiian.7.7o suaid 2.arirlc Z (t1':a 4 I t ha. c durirn E. tAu- i.: inrL'ec-

t ion the .roiuth of Cl niri. i iim r ',, .--< i. i c i ,-latc-

tunlle -'Otb f L ,act. ."i' lii :p. i crir .:p .--.I The.

,eca-l L.i t:_-ri l 'lora ,c.nr ti .u.:cc '0 uf Lth- tol 'l i3t'o-

initestii .l filor'. 3n.9 is of' *r.- i .: :1 i. ':aI :. t o.

th-: h:altli of *ci:,; ie', fCor :..'nlh 5:V i .' -- L.-" i. *;i :.min.,

ICo.'t: : rt j l., !'4 ; Tinmm Ir .-')

IT e c "-r 'lc t ,cf Llori.lj) l.a-c : 1.1 l i l- r; i Cu bi olo '!D :i

ani irr.nriol.';,' of the ho- .-.ni ro.il.le i : at :n: ip Lb-ticrn








this flora and certain diseases have bea-en inve'ft ig-.tgei by."

maLny uorkei'L. Pnillips Et il. ( 19.. rising e-rm-free

guinen pig pro'.ved that pr'esnce of bacteria al flora is

Ce.ent ial Pfr c-urv'ivzol of Erit- rlno' b' hi. tol'i: ico and pathio-

jecncciC oir aornmebias i. Bared on theze findings, Wlit -ier

?-nd Rozentbnum (1970) ctudicld the role of bt.ctcria in modi-

fyiin the virulence of E. histol'.,'tic. Bradley: anid Reid

(1l966) demonstrated a dual etic'lo;:l involvi' zi a proto-oan

(Hi-t..r'on , m.!e:.'ridi ) :rLinld a r-i1ngle .pic i -es of bacteria

(Es 'cir'h'ti- c -l i, C. nr r'rini- ns, or B-ci ll'ic ubtli s

for infectious c:nteroheop-it it in ti rk:c'.s. Several other

studiec hTi'ave been inade on tne role of bacterial flora af-

fctinL the course of infc-ction in infectious entero-

hep..itit i (Doll and Fr,.nk.er, 1963; Franker and Doll, 19?-,,

Reid ,t! '_.. 1969; Springer et Al., 1970). Hejde et p1.

(19o9.1 studied the patihroenicity of E. Brunc-.tti in t4actcria-

free chic!:ens and zhoued that the para!ite can develop and

produce disease in bacteria-free Chic!-ens. io substantial

studies, hc'iever, '-i-e b-En nmaie as to the rposible role of

bacteriia or other microflora in relation to patholo;ty due

to ~. tainr llr usinti-; ..outobiotic i'bact~eria-, f~uii- ?rid

i.,lcurc'pr' curonia-li!c- or :-;ii'r!ns (PPLF )-fr- ) chickens. In

pre li:-. inar1y stu lie", Clic. ct *"I. ( "..) f id no .liffer-

Cnce in the course of E. I ,n.:l -, infection in b.acte.;ria-frce

and ccinvent iona-l chicl'. t''-r'c -. hone'v':p a dela of

1' to 15 hours in tl.:- -ippearic-eie of the 'aiJ g:fnerati ion








ri : P::o .t: ii t n ? fe,? of -1.:tobi:,ti' :a L?; V 1 fc.: :nd

PL- i.t i ( I ':. qi.. .:.1 .,' He,;.i: t l.., 1'... ) t e, rt. ci 1

rclation.hipn .et ': ce hlt- micTiil.P lor'a .acn -. 1. in

t ,e l:r-dtc,:l:ioian ,:cf rlHe :,.. 1. *zc::.? ,idica i .:i -n.'l,'i"r,'c. .Ac H -rie

Lt li. (l ',':) rI t ted t i i'ct, c 0f th Lb.act-i 1 1 f lor'-

?n t e:- pat: n n :,_ ten icit of t hi :p c., i- e.- .PC 1 r ,it.' I.Tn':. 1 'v .Ji."

It i. nZILOt leA I he.-tlier t ihe b-c terial fl-ra niri.l2I's C. r

o1i, '. t.incll-_I to initi:t':s and d*.velop tnhe 'i, -e c:ntity .

Tt,,- przei-c"nt -tu, dy uw-s w.uvile rta'e-n t c. find out t the inIflueInce,

if anii, ,f t r e riCt':'Lb::es normal r .' ::ent in the ca.: o:l

chici::. in thl *e lcmnt of cecal *cccidicsai, cy

I,:-terniniii.-l- th.r l li ty r.f E t r-, .- 11 t:, pC r du 'ic t I,?

specific rritli.cv and legion.; of Cccil CC:-idioSi in

:'1ii,-:n, is i bl..:,'t-i no :, ,lde,:-e, able rtii .:,.r' ism :i.nd by

cc.rp.rtinrig th, pa-thc.lo of : :xperim it a':tl infection it h E.

te- 11'.: i in Act t'ia-, fu_,,i-, and FPLO-C ft e- clhickens,

p:r ci i pii t:]se In- r : e ci -1 eins arnd c :! en lti.Y i O al 1 c i.:un:.

T'I:. .pr e i;:.:l'h po.-;late ith reg rui tc co: al Cc.?ci io,'l,;.

r ct _r.ial c.1:,1 't: r ':r c ui',venition,. l ai:.-iae-frr-. c jhie ens

--nd chick--ns :.. 7hinig typical l c ion:z c:f cecal coccidi,. is

i- ,r- a- cl:, c.p:i r,',,d. .ite 1 :i fu n i-i- :n.tL F LO-f roe a.- _id

SFF chicl:cni 'icic incci.ulat?., i:ith .ta n.il- dose:-c of iF.

to 1- -:l1 l :r.: l1-it-d o. K ,c /. l.,, 13 K r, I O na !u.r ll;:.'-i.W',C. ed

ca' e:, eith .r ailoie r ..:.I: .. .L th bacter i-l ':ci- s.

Ar.- ..'t" ai L c. i o'" .. i tc: i of -i-.: .ni. : c -:i bl of pi:-

dui'iL 'e.' il occidi ii: ii, pi' tidtoI.iot i clhi,?:r.i wI ere tSn ln








isolated :-,l tihe i:. 1 ltc.- used for, further inoculIt ion of

conv;,i-.tii..il, SPF and t--cteria-frce click lcs. The l.nou-

leaugL' of the intci'relation-hip betieeen the normal microbial

florets or the co.-ca and E. tencll b ained by the present and.

subzeqiu-nt studi.-sc ii',' ult imLatly lead to better, mean:i of

control of cecal coccidio;is.














LItiCt-iA L[Z~E RE" 1EV!


E_, li- tr-rell-i [Railliet, and Li,?I. 1L'1 -. 'a

prc, tLc.-.n t,-l, ,fin t'c the F--,Tuily Eirm,-r iidae, *Clas

Sar : c a. 'i'l'i- tern .cc.L i. is" i. g nce r : Lil sc..i t to de -

c'ri b? :pec'ie beli gin; tr. the F ril- Eimrn-ri ida' i Bc e-.r,

1'.4 *). DLurin- the i..t 10 y Jar t tLidies on I;he fine

:trltr ure cr f c.,cri ..i and related crI'o. upi i._-.r rc ':*ale:d C T

cre'' t ntr'.atcr cl,' h-, -:imil'ar' tr',r tuieU c namel;, the :,,.llicile,

the rF. Lar riii;:- tie otp.:11iiiiCLij nicr;:.tut-.ilc tlhe

I h .':-.; ie? t'e i C ) 1 Iro 'in- the i er rc '.',:- and ti' cc': ,.

Tle.'l fine : ruc tural imiiari tie re ie : c id-e-i r:i d aI

indici aticr cf a ?cloe: Ir: 1 t, icnr-rii p ad Levine I 19',.) pcc-

pI.l d a s ig hlt i ood iiica3ti.,n o :.If t his cIlas C i cicaticn iand

Si: 'l:i -'c : a7d -l (c1'-70.1 h. '.- ,i d Zi Ci ..:1 tI) pIr-r...tbl-':ms

r'f t '7:n'.,in,' c'f Sp',,i'co --Ca. In th-e d c'm es I .? chlic'en t'3 11 Li "

drer...' ti,?i ), epr:-c les f ti-h.: cenu.I. Ei'--,l ? E. :c ,rv',j i -i,

E h,. i, t l- .. i-_- P. t---" ini i 1 ti- L'. r."j -J i E- .

..I-..-.-' ir-, E. -i ---c -nd E . an-ve ree7. n d ..' ?!-i e

ra p.e.- sites of r I. -oith lial c -li. f ti -, ri,-u i.--iCn.

c.[' the iriteztinml t ract ',,,iest-. :r.J icn.am.rt- I'.-.. I. E.

n *iv:ti is tle :il' cpe c.es ihicilh ri;,' b: f.und in v.-eral

e i ,r.. c ?f t:, i t-. 1in 1 Tr 1;..'t ,j".ir :ibcld, 1' ', .'i

T'ie '3 rpi-eci.- cai r di fcer. ti- i t -. oirpholr c;ic'1 charn.ac-

teI-i t Z ?C :;-or lati on tim: ci th. ,';cc';a d,;,v,'.rl..nent l








featur-es, localiz -ion ini tLEL hoct, inl deg.r ce of pathlo-

*,.pnicity. Of the mirphc.logical chr' ct. r-', I-c t "ucLtur

-f the occs-t i: uaLj-Llly' u:icd to tilntify thi pecir:: at

least ithin a ri v.c-n host Lc-'.'in 1961), but oocyst

c aractcrs alone 1have only 1 ii ted va].-Il in differe'' fntia Fin,

pcc ice of Eir-i:'ir. (Horto.n-Snith iand Long, 19(3).

AI.moncg the mnii,' means of biolo;-ical diffcrcriitiation

for Eineria s icciec, the location of ;nd'.coge,:nouiI stages in

th.e pacific region c.f the initcstinal tr.ct of th hIost

and species specific i niri,.it:,' atre of r'3j-'r' importance.

Each spcci&e shoiu a mar .ed regi'-onal specificit, (Tyclr ,

1929; Ty',er e t el. 19?.; Heirick, 193c6) for the develop-

ment of cindcgcnou- ct ages. A lsco inifect lin ,ult h a cpecio2

r.-cuilt" in immunity cigainct thait s'ci,-'.: butt rnot against

others even withinn th-e .:,sre host. Hence c'ros-i:. lmni ty

t;st? can b:- used to diff-rentite the various peciez of

coccidia (Tyser, 1-9'; Ty:zCer it rl., 1932.1 but tne

specificity of acquired si.-stance may not be rigid.

Rosc (190.' I) found: crors-i irilni ty b: tx'c," E. tn n ] a and

E. n c- tr i ':h-.n -, ncirg pcr'c. Ooi c -, of E. n' .- t i t tc

induce infectio in lie c.-m,. '.. reti ui'i Lr e a .'o-nbiration

of factors is aI l":.,l u :ed f sy- i : if-.nit i icat ion.


Ei ei t enel Life C i : i i.' .-li '-i: I--:

Acute cocal cocc' i, '. in '..3u.v'J ,'li,. .n is r'e gLu-

l rly" assc>Lcizated witi E_. ,- ,.'' _,d this .spe..ie- is- tle

:no.-,t comnion -aind r.C : patl'c.enic cf lil cocci.li found in








,icl-Ini: D vie:-.s t a ., l'.i) T : r (1- 2 ': blilhed

: .1. tai e ild d.c',:.r pt i:, of :.t[ihe r-,lr'r. ,lc.,-.r Z'.i life 1c.lt: .of

t. ten 11a: '.,i.:h ha b,:.c n confr.irc'..l in all detail by c,.b-

.i:ql'nt in:.' ti; ti; .:. (E.iint, 1911.' Lii!:c other piti: es

Io EL ri ic.::i. l s' .- c l : 1 ge-lr at i n o?:C.ur i the-

2. -e host rol ,lo 1c An 7 ;,,ce-:tin of' v.'iab :- -:p :rllat :-. -c: :yst:

t r,:i. .i- t'cd ,.'or ;:, ter-. T'hruich a pr:oce:. of e:.:' at a-

t ic.n, sp r'.:oit esC: -. f. ir oi tie E .-rc.c" It: a.nd ooc/ t' s,

libut th the: f ct r.'i co rt tI'butI i n- to :::;- stati n hi n:ot bee

d firi itely' *:-sta.rl i : i.: Stl die- b:.' Lrvine .19' ) and ,

I e 1,l'.5.5.a 1&55f;-, 1 ': ., I'.:). re'cale,1 tn.t pan:r.- tic

, i .e, in part icilP.r l.r'p:i is onr- of the fa tor: r:-

.ponriLl for ,::c: st.at iocl. G,:odrich '. 19' .' ob -e; v d .

e cc ape- po : i thi o i lir i; I i .'.ai lab-6 f act urc in

th:- c t u-li 5-10 m nut.lll i : a. l er tC. C:, 'Tr il has: hiC n

phi .d in a 5" t r./:-.in So Iut ion,, mat iit i e:d t .70C

H:',.I r-o ni]- 'i n con:. eri'Lt' r ic', bi l ara d buff"e.rs .wer.: als,

url.,nd to be in l ort a t factor: in a::'.?,'st tion of va riou:]

:ppC ie cocci ia 1.3S tana. 1 3.."', : Lo.t-e i1 L.- -: I -9-0;

Deran and Par., 1:b2; ;i yb- r in. d [f"li.O d, Q l':,'l Hib.eL rt

- .1. i.1 t, :tul:,- i r th. 'eci. :. H, C.f r., bile

-nld bile acl. .,i t :.: ',1' : C -c" '- ites of V 1' ri

Li n'i :p'?e inclilin .. i 1' i L 1.ind n c :.'.: l'-.tion

lih,:i l .--:- Of tLb: bill: -aci,: oi : ,,.i iT,: ..:.r ,:ci. rl:an bi 1: uar

Li'1 il 'iie i itiiout ;,r' *i P. I 1 H *r! tte c- ,:' r d F-- 1- .

t.i n C" E. f -'.' anld i. "l- '. .' iC b-V. L i.e .il ,: : -

ta i _i n a h r,-- ." p.r 1' o i o' b I:. .'i. d I'ui:-i Iilien








trypsin alone vras ius- only ,. l.ov i jocysts excy:ted, the

other 9? secies of 'im "r i- iImnluding I 2. ten, 1.11 did not

excylt. The precise iay in which bile actc in e::cy;tat ion

of coc,'ct a i nolt. kno :-n t pr'c ent Doran and' Farr (19 62)

sug':c:ted that bile acidc m Ty laterr the pr.otlein or 1lio-

rrc.tci n surface of the zLcid.-i body in zuch a way that it

is thiicn eadily nacLed upon by p.-;cri'atic ,ncyime. inl/'or

mat facilitate entrance of onlymes into the int at oo0yst-

throu rh th.'- alt e '-ed micriopyle. Lot e anld Lek: (1969)' found

that in aduli chlicl-ken.' ab.:ut 40-,O minutEs may, be required

for F. tnic.1 a oocy sts to be : iri 'ted from the jiouth to tlhe

large inte:.tinr-. A Cper-:i-mnt .Openinr or micropyle was not

otGs, vecd in thi oohyvt ,:all tihrourh :'hiclh poro.cite.a night

eccarme. Tli-rcfor' the release of larive rinumuer: of s.;orlo-

zoites into the dige3st- iv tr-ct of chi'.l;Ena requires th'f

rwall of the oocy:L to be tbr l:.n, Iea!lened or partiially di

solved. Lotzo iniid Leck (1 .69') observed the ,.all.-; of nany

aporulati;ed ocyst e:peild thrclti fece- to be structurally

changed. D.-evelopmint oCf iimmrruity e not hind-r e::cy tn-

ticon and nrnir.al e:-.cy:tat ion :ill occur in innriune and non-

irmituie chi .':en' u.'n.dier *:.-tinr l cci:li, lon5s ort n-Snith -;t .i.,



Li .r. -. 'poro".-' tr- .c fu ifor',-, IC n :- 1.5 in

di-ricter, tra.:iisp-rant ,iL' 'ot il:c -ch 1 -oroe ite has a

uclo -i ;ro-. ail .t .".*-.; i I .. :-I L Ile *:it the uuti-ided e nd,

anti e".libi '.'ri-'u ty'.-.. of o O'.'cr:n They r:i'idly invade








ti ." l:trfc':e .-pit lii CAR:1 Of- the c :'c i ... th.t.n. pinE:-

r t I' :l ? L i' : t n nr-:-.:--n.- to :r t r e: pI iip ri

tir,:'.i'-i i, .n th'l', --ip hi-j payr ,'.:" fr '.- ,ri i i in tl'- ffiC.,cro-

pi -::, to C.lail reac ii .he :pit l isl c ll liniii' th.

fuid':-.C tone Liabork-ihu :nQdF -mmr- :':uil in rIPx oducti kc




l -,. 'r* i.rL. -,-; '," ,: t ', r i'. .ll iO]:;, ;l hi, i.'-t I: ; i t iml .tr




.\ithin :2i.: I l z. Thi ,ni c l.c i-.i thi invr. d call t --

- n rI r t-_ hid tiLi-, l-- 3) ..l thc r: t IO L-d

C l .- :.i o ut t] ip b'.', 1-, i E" '. .'-a icivt f' crl

t i.,.it 9' '0 lr- :: oi- : r" .-z:r, t1 t"' ) ."-' p i'i 1-:t i-t 1-a

in idi,'t i. -:, '. : t.: .=i ,- t.' r- : le- .. .: i om b ', .ire le 1

, i ..-io Fc ii i tt n t- r a o Iiin.. I 11 y i r-:. p-;r-i-[

t _at-l on ii: m- i fl jlr) -te n- t, i;:b'L-:.L iitli-.:1 1 ,1 l r: f,:[ t,,. ti.-.eu

t'. 1'.- : 1,':p a- t -, it geli1.;n it F.i .:hii:ointil:z. T''i .ii l ultr : r..' I ,.ir-

:,f .- m r-.-.c ite : lri.- i the i i ict ii- :j ( c c i.n.'c have beei

describe!i Q: [CLaren i1.1 F- m A 1 i ,.:.. 1n IcL.arc l u09).

r )1 ii of t i1-. il i K-. l' 1, ..OCl h i it 1 1I I tI a'id -. t in

- C h iir r .I rc .!I *u. O Z -:, c :ii i i-: ii : :I' O i: E- ,

c: in o. con:, : %..7-. T'he r1. - ',. :t:.,-. .:ii .-,. .? ,:f I : .

.- ",-r i -a r,' fc'J in t ,-. it .-l_ *1 ,1 .L i t-'-.-:-fr I :t

iav'e r':,,-.l fro:m th- e .- r i t:,li- -1 lI ::.-; .n o tl:- -:ui p: i :itr i -l.r

lan,' 1', Sij WOZlC 3 .1 : >0 ut --i -all j4 l- 1 .- o- the

C .A. T_ i na .rT .ion An1 :i. -: .L V L )r c I ib : o' Ca l








clnerat ion ner.--oit ez causes extensive dcttruction of the

epithelial cells an! severe hcnor'rlige occ ure into te cocal

luw:n I C ollclocd by tissue necrosis .nd thickening of tne

.cccal iall by the 4'th :ad 6tn diay. T'in- 2td ,generation

,ro.jZoitezs are considerably larger thin the Ist, 'nvcr.iging

abcut 16 p) in length, 2 p in wi-1th and 2.00 to 350 in number,

many of ihilch cntcr new host cells ajnd begin the scx:uvl

pha.e of life cycle by developing into uit.lici ncrof_'-aeto-

cytes or mricrl',jaetccytes. ;icroga.nrito'ytes .r'e smaller

both in size and nu.trbcrs thn.-n mri.lcro .icLtayt':es rind the 2

ar: fou.in iin clo-e pr'c::imity.' within colruna r epithlielial

cellE of tie ceca, belo- thc: host cell nuc.-li. Fron each

inic'rog'rietocyte nicrogd ctcs develop. Each micir orncte

lhis 3 fl-Ta ellia and is notile.

Young rmncroganetocytes are large irriegularly shaped

crlls mea-uring appro::imat.rly 5.3 pm :: 7.5 ipm. Tho"- '-e

young macnroP;n-etocyte still retaiinis L heo zhapc of the ..r',-

Soltco, iL can cbe diti1;'inguislhe-l for.! schizont or micr.c.ameto-

cytes by the pr:sr.nce of "wall forming" or nieribran:-ou-

bodies I.Scioltys eck,, 1 i' ) un..1.e thic -electron Ficroc. cope.

Latcr l: bodiic-E" d,-velop chich are t o'.Oruiht to corresponki

to the "plastic L' 'nilc s" :1. u rr'ib: 1 fri -- li -t LiicrccopL.t

inve-til:ation: (Reich, 19'13; Dofleln a'-. i. RcJ- enrio, 1953;

Chei3sin, 158). After fer!:il1-:rion these bodies nizrate'

t,., the p.:riiphl,?r o'f :y''ott -. ii li ,it mcrLran,: of t

-,', ote th'en separate fro i.n the cell to bt-conm the oute'rmost








Mei br.t-.tln- -of tli c:.-..m yz trill. F':c ,iiyIdl: l r.ye- .Ic t'r o cyzt

--ill 1 : -.eicop fI,:,ro:, t "d.ani bl ,il- e an.i th "ih Ill

for 'in- b'.liE ." iv. iZ'-s tC: ti'e x -r 1i':;- ,:r -of the c -. --. t

i.li Tihus, in E. t-n--~=1li te oc,:-,..:t .Il i i" tr il:m'ante.

'lh:C- th-,: oc.,:: st .-.all is .co rnr -, tet thc ,:,:cy/st. ic :: [t .e.i

f['r-n t' ho: t tizu46- -Ind is' p. :e to tic ::teri-, ii t

thi -. :-:.- Thi.- p.-rici f f vr mA tlIh t riie of infecti c O to tie

13;. pp. ri3.ice of oc:, : is usci.'li: 7 da.:. T'h. C:o-:,s

product- ion th l.i:e c.rir'-.",cc:,- -O tih-: 7 'thl i 'J Ioll ii, i'- Ce::p..,s.t:- ,

-re-..:.l z a -'r-:',: --.v tlie c th i i a a.i i. r:ipidcl,y d-e :r ?- es..

,:.:'- t-. orf nr'-l1 m c0.:-i-:ll, Ecl ai. ti-:.i pari'-:cnt, withl

Su1 11 I eI'i ,-Id dOWCubli Out lii e. Tile ,, G :. r -,1:-. r c-.f t I'e

o,:,: t '' ill w; ui.1 n L- cl'-u L L rot i anr.'d th i luier la.uy r

is lipit c-.?o-t firmi:.' .:: .?iat:1 uith prorein larn-lla

I-:-lfb? .ni d ii ., 1 ': 'i. Th ic ofI- t -i c:. c O zt s vi-n-

from 1i p to 1."I :p 9.5 to '.8 ., i, oift.h .2 ti-can cf

S.':, ( O. 1 .. 1 .1,-- i, I. c.' I :i :er: 105 .:. The

optnimunm temperi-tir--: foir spc.'ul;rhiL i of thric.ooiys- is

2.0: 7 1'-' C. I.Edlia', 19". ai.] :t tl: i re-pe lra, Lure -:pomr 1 .l 3-

tio.i ,ill bi- copl: tA l -I 1. hcur: tbitt N t r:.-o teripraCt .:

it ta':: out 'i3 tour:. The porul-.e. Dc :, 2t 'contai n-i

4 -p noc-.'-t : achli :ontai inin- 2 :.. .. it Liln- in othe-r

' ioi- f: c ,f Ci ric nl.- I t i 1 : I '.. ii A n. r ..: lc" i .

p. t-'l 11 ,-.i 1- ct uit i tA.:- in' ti: 0. -.; rop n chic.:k .mbryo

I L:,n-, 1" 1 3:-.. 1 't- 1 : '~i t1 .: cilt.'ur- .:. ll

(Fa.Lon,i 1-.5 P ':.ir',i!:, 19.'. I''. ; rout N- r. a O. l i te,








1''-.; I ;, tsuolck-i et ki. 1t ''0 ; Dorn.l, 1970), using rp.-.rc,:cit.-..

obt in l by' '.t v trr, e::..yct:t ion.


Pthc-cncsi of" te"'-11L Infcction

F.-ctors -ii acting p-thlropnicity oF t i-n:. in-

clule the nutmbr of cocycsi inreAtri the nur,ber" of host

cell: dc trcvyed per ingested occy,'sh, the dieg.r of rP-

infection and the ztate of imilmuit/ in the ho.t. Thec

severit.y of the disease decenjU uoon the interplay cf tihel.

I.nou.n an.1 other lnl-::no.nri f'ctorc a.: li I'anr.' I'rc'm a impcr-

ceotible reaction to i.:ath (Gardincr, 1955). Cecal cocci-

,di-;is unier field conditions occur. pri,Jcip-:lly in yotun

chi-.;:: nz b but sell. on in those l -s than 10 or- 11 'dlys old.

The range: o1' cage of' s'usc eptibility is froicT 2 twieehk to 1

.int .h ;Ml -I of the wer t out breal:.t occur at tl 'he a e or 6

to r.jceks (Biester and Schuarte, 1'65). Herrlck et _1.

(193r.; in -i t. ,y on experimental inf: tion I-oun tlihat the

heavi.:st mortal ity anm greatest d-l,:creacse in cry t}nocyte

o'rcurr'Ad in chicl:z 1 mDnth old: heavy mortality -lso occurred

in chli!:s a.-edl 2 .ec::S anr 2 monMtlz a-lhile in Alder birds

1.3, 4, 7, 10, arnd I 1 month o h: .,c i nr' .-r alit' ... lo, cr

lack.in tlhouli drop in red cell courtt t nel d ir..-m -'9' to

,'.. ':. GarP.in:r 1 955.) e- l ,oyil .-: : ~_. 50, 000,

100,000, an i 0,.000 .. p'ul a t d C....C,'. t iitfa t. yO un

c;',ick -ns i. r .:"i':e groups or 1 4-, I :. W, .-cks.

Those iQ- the 4 -rup r !].r ffccl:-' 2n.i t'ho"e

in tihe 2 reek rl'cup i'rc 1 :-:t affected Thoi'..: bit.s 0.hich








rc_ ':-ve from nif -_ct i,:' b:ic.:'.m': iiL:- ie to r.: inc :ecr in .ith,

E. t -11 Hoev.'e', thi' is .1.t ari ab c'l, te:- iinurIit, .

.oi.i r *:' n i,.:n; ,' r- s, t he- acquire r d irmniu.nfi I. 1 r ':ti.r-

oi rdz r,-y b'real-: d.i c an- i -. ;,'mptcn, of th' di af :- to

re ipp i-r (L.:- i.n.:, 1"*'.). L-vi .;- (l' 0. in a r-tuty ;' .?ub-

cl' inical C:C:" i.i'l ii'- i i, i ull,.- : at lea t rr: m ntthi

:.1 r : t:-.l th- r.re ne'?.:- : E h 'i 11a in ': '. In F:.-:neral,

nic:,,ver, it can 'L.,:- .un i t click ri i.::. lI lated -.i fro

iriC'.:- t i:.n remain fully and u.i.' rr'. :il, u .c .pt ibl' t hro. Ti.r j-ut

tn-ir live and. that a1 r :' se hc ':' inrl'.e'cc- on ren ist-

._rn:_. 1n..ier fi-l.l ,:,.:.n itio -.r aln.:, t alill ,:l'cn! t n ,rei- early

::pc,'cu to' at least ligc t inrf:-:tin and c: nCa-nl: all

c ide:s re,: than .:n :- a" :.l. Have c'e l 'r-: :- f r is t-



Inhcriitd reI :itEr: in C*ic r tri: tL' ch.lC'ErrVn tI

inre,'t,:n .i.ith n te ,.--13 h 5 b1.:,:-i r pr.:,.r ':- b:- riosen .-ilr-',

I Il 1. n. Ra,:,.nb'r- ., 71; Gibb-,_i l .?' u it I I ,ac-i-nIr'aI

th erc i li jI :t c-:irLei I anI" ti Zii ic:a t va' riatl 'n in

.r_. '?, ptibilit 1 t:e., Ltir.'.Ti ,ii .ffc 'cfnt bre .-.is -'r sir ,in. 1' --

hic ': i : : I H.,r- c: Er. mit r .n.1 L,.|., 1:3 i J f[r: andf

'.':I it i I I..'' reIpIrted Ih cIIr -i ti ili and i cr-

t .ality of r naie c:hic:': -mbr y.' '' ':., 1 :1 f.r nt g :- t ic

zr].ii .-:C2 to 5. t:.- --1 i. inf'ec.?ti :. Li.-:*. :.j..i H,:r, ic: (1 ''-'1

pri-Xuci? cviei r ti.c .I:c.:l t.hi -t ri.- r irezclrc *:flr act in the

di'-e:ti'-re tr:i ct oE ci rds it .-: ti-.:- lI inl C'l.i rc:-.1i::-

t1 e --everit 'l the i eis':-. Hcl -: er al. 1.'-' :'7. .i .e: .-








that inc rc e,.1 r- it i r nL nray reu) lt in chiol;ern hir.ing

hi -no:r m.' n iount of -y' tcr S',Iell in th.- ratic.n. IThc niir.ibc r

of oocyC t: rei .sillt ing f '.o:i n inft'-ction i.. not n ti ue

inli'-ltio.n of the de-gree of infe, tion Tyzzer (19220

postul1 -rd that, t heore tic:rilly, infection iiti a s iingl

oocy.;st or E. t 'en l l.-' crc.ul1 gi'.0 r :i:lc to .niproximn:t.clv

1 0C, ,,',00 ooc,:st7 in a period' of -' to 5 drays. During tlhe

co* rsr of an in 'i;,: t_.n, hc.i,.'.r, t t,:r,2 ..i .,v ra,1l factor:

, ohich r,-i." cause c re lu.: ticn in this pr'-Lentill inclui ng

loss of mcrozoitcE over cr '.. .ins, irl tiscie darii.e which

results -i a los- ofc suitable cells. -r:a..':ett i0d Blinnici'.

(1949, iim5.. o ri p-rted tiht for ea.cli ocvn t of E. ten,-ll

inoculated in light inf: tioi ni pprox:ci:.atr :; 10,000 o tcysts

are~ prlr'c.l ed-, arid th.'.rr ic no direct correlr tiion be.ltuewn

the ciz.- c.f infCe.tive'.' 'e io.-o the final di :'ree of i11

tion. Hoev-r, J-nnsiii (1927) repr,.'ted tha1t thl. sE.vcrity

of cecal coccidioi is .l-pe3il on the uni -ber of zror!ilated

ocO'v:ts tLiat thi? bird t ceive. Jc :i.i:iic rid Scofield

(1 934) repor'tAi th't a dc.-: .c c.f ui. to 15,0 ar[o.lated

oc:c;,'st: rodn! ce neith-r vri '-:'s nrior' rL:'rt'i l it 150 to

.500 c'.cys.Is pirob. el : ht I,-: or.i-'e Ln no: rnrtalit,;

1,0'0 to -,00, c,.:.:.' t. foi']. hc.' de ir of ie-.oirhcge

and r'J.er.t- nort lit ( -d :.'r .'. : c.in .-. r.p' eiu he-d

.ae-vcere liherorrh-;-. -'. ii h ,: cr ality. 'Iih prepritent period

in E. t ln.- in.if',:ti.o: i ," '. ',... Lih p-,tent period

v:nie : .'i h in .li iduil ij[ recti...n F'P -h t3]7 31 i r-pcrLted








ti.::t :,ccLr. s ier:i not Fpr -zent in the .\ro. iqia z. of the in-

fe:tc:d L'Lrd a't-c 1,' day,-r -ith u.i Ty z' r t 1. .

re-,,x ,i *C :' t pa t-.-' *o r ? ,. l i_ 19 da.'" ri'it-

in .cct ion. Tnc ,:rc: st-*r i'i-ib rs f c .:,cc; 7t r di.cihar-g -1

in a y'" r, 't tiy 'r,,-: r A- '1.. 1 ") t, th: t-:: rc-

13 ,a i cni bt' n'- f t I rl rp.e.1 it l-:r in ti t isz;,Si. e 3 i. n th l-

c.-.: i .,rtint c it:; ,ti rre-,lari.. r.-1. :.c i. U i -i rntur- l



many,' r.a oo: r.: Itfor ln perai ti ,e. For .-:-iMple.

Le. -in-: i '-! :) .l-i :r .:d a:o.:' t f: !' t :-L 1 il : in the drop-

pir-s of out of :0 bird; : i cich did not i:i an:;, mnrpto.:r

of irfic t :iri .

-he dii -:.:-.-: -.'i'rpti-i: i.n C:.-.al C: C: : i i:,c'r i .- '-: c:l,?':-ly

r":1.lted to ti: o: ur.'-: of i'f cl, io,:n a;,.d in g-era'' l tl

d**r:- .'f p.:th:'r ni:ity i rel t.:d t-. t l-: d pta o r;,ich

th.. c.- l iall ., ariti' ed. B. te -11, p-'i.:. ra.

,die pl:, jL.i i "C.:r' d,.i :.ilZ t ti t Dur in I. the dl- el.eli :n:-i it io

t-ie ,ar':_,itr-. ther-e is a mni,*pratic -i f ,ara'iti- .id cell:

i t the .u'L: .ri it.,:-h l i al -ii'n h,.er,: theya i:icr -e:-C n:lr'i,..1iisl

in c \- :.uc t.i I ..-e 1: d- tr Li d and t l i i: oir f n C.c .

o:,1cr '. ,. -: ,h: i- r,. ,:.]f r. tnirt i.aati .-i' id .er-nie i c:,i ," ii ,:riint

3: ca' 1:' n. t 'i '.tt fuijr el i .f ti.- :n -n .1 of.I'--

enr,:,r i c :.: .r du :- t o : h.i .:- 1 .. 'e t. the bl:.:od

'c :1:. Thi: l.. :--d n.i- i I lt rio:t i. : rtL ': : t f.r -the

r-.-ra 1 r t i n r. i:,ort -Uil .- i : 1 l1:.!; t .. .. I L ho r o' n:.

-i-il conrt ,ii' oizi L e jrinr,- o:c r.i s f C rrn th : lth to 'th ,t '.'.-

p t -': p o- . : ch of th."- r, '". al ." i' : c-- rn








sc-.ondary rh c i ..-l 1-l i'nfcction of r1he ..ei' ii L which the

ori h.llim 1.: l completely dJeztro;,'ed lEt i i:J I, 1 ):'1 Hem-

orrhlige is a gre Sait s r'esa oan the infected chi ckl'n and

feeding andi movement riire at L rMiAininiar luring this period,

bLt con Lumi tion Co f r'ater is iiic i: sel 2 to 3 tim e-- that

see:n i., u ini'cted birds. In a typical zeverie infection,

bloldcl' droppings x;ill occur 96 nourn pcgt--..pcsirei and

pa'age. of large quaintitics c f lood in tihe droppings con

the 5th aind 6.th day post-exposure occI.;r. The di-:e- i

at its peal; on the ?th d .,' poF .t-e-:po:i're anl W '. of the

mortality occurs withinn d-ays follo.:iig initial 'c:':pocjutre

to ocy,,; ts. Chicl:rios rlurvll'ving 9 dayv following e::po'ure

ill IusZallj recover A chronic condition', l io'w:,' r', may

occi.1r ?as tih re".i.lt ci' retention of a. core of necrotic

tissue in the ccwa uith consequent ,'c-lA dy .runction. III

the flrci: as a Whole, th' diIeasie is rrarly alaci:ys of short

duration. It is often fouid that a i-cndit ioin cf dizeeie

arizes only rhen infetion, he i vy in ,elation to th. pre-

vi'OLl. e-xperienco-% of the birds, i-. acqui red during .a period

of 72 hours or l-,s. If infectinn is picked .Ip Ino'- jl y,

thc.n the bii'dz c OILcOr re:..it int l.?for: cliiLy:.' 1 fect:

.-ppc:'. 'J?n chic:lken n re r- i .rl n ep it ttr D' s i i most

parrt.: of the icrld, the ooc. .ts ar .t :. .-. ly dl -' roved

by th.- heat of ia -r ent-iat ion, LLit I i o:0 tih u1nfavor-..ble

enr iiro.iirlnt .ire pr slmo incir'- tly v nI'-p'rinli-L 5. Cia ~t;':.-. -n the

crnv rocr.ient, notaIly nn incre.- se 'i rn icstjture .id -'r te nperat ure,





20



fr .:, :' hi !h nd r..pid rate .:f :i ru.litic.i l:adin:_- t..: i

:liniC: l di.e1 in thit il C!:..

'.pp i ri: ':e f i e:h lc''1 irL th:- d' l:'t.:.,,i 'V and

s.dei. ni de, t. h i r1 of di" a;--n t: ." '.'31 ,-" i n ': -i: -Il ,,:,:..:> ,ii:,i: .

Clott i :i,- f lO d L pre'- nte i .i' r i hg t :h '.i : .S$!ez t-,'

zone n kniy.:-:,i. ri ,f .ct ..,P c I'. L v i t l l':., mci d i,: ic",,:i,:,

*:" '.'i ta i5 E ir :reL e-- patEh,:.t : iic:'it, t.: -.. I.-' i ] .jri ,1 E

r;:, ,t ri:. tLD:i'ies et ,'.3.1 El:.,:d dp:,. :- t, :-::.*-

pric:': flr:nm Ph- -nt. C:, .-T-end cir :.'i > g '.i :.1 up .itlint- --, t

nco,: rl-.. ,t n c:lcp-,: bl..:.,..-filleId (..: 3 ,and r,-.:-cric- ..:.i

de, elc.pm-entai l .:Pa-e: cf: E. terll c11 : .:cnfim- ti e .1'- ..,:'-

i.- CCCC ,L. c, -,, i.1,, . i n,--rep gfre e of .C,, .. r ,

nrt indii:C i of di-e.--e .Lin,:.- .n 2. t.-- i 11 ii-f ctl ic.nr-,

,:,-: y ." r.: r.-o .:t ,)rdin3rili,' zee-n in a:i infectiC:,.i 1uf i, i i. l,'

e:: t_- t-:, c: .. e disCieaZC .rnd de-th iDa',ie.: et -.1 1'c.

Fc:ir :xp.-.i'[met,;al infe:ti.on,; of c:..:cci'i.] i: I:nc-.oin

nru'te-rs c-, ..pc:,-'urlated .',:,cc,'y t are a in: ni-riist:le.i:d -,iall',.

H- .:'. '-::r, Da'; ies ani .-,Yine L'- I, -'o' l .1 ,,i ..ha t' atnil F,, id .

Ei rceeded in pr-i'.i.: i C& :al iniectlions b',' iPntr:inii tint U.

t'*:- l:'i- occyst: Sub.ut.aic:,.i : .', iintr'a c--: ;,-, iintr -

p.r -itc. -nc:,l_:: or inti a-iu :i1lar-i-y in ,ic' .'i 'inct i -. i l.-c

.:pr-..: :ite- u:r; intrc liiccd b,' thre .r-n-- r-..:-t3, i: t-:d in-

r':, ,i; '. ,:' rc .,'.-*- ,I d. TP : te :d ,: '.at ic:-'- a nd

t-ran rf-ir' t:, th- oile c.if Inf i cti n f.I c f: ci,: ,. .: i.-i

i n:cc l:t7 i. p or':r t-te.-all: i- n-t ', t ',, i-:. t .- H: c.rt n- -r'it

P n.d Lo:r- ,1',. ) ;cn ir' r:, th e.:e fi.rli,.-s p:rt i l 1 ic thatit








aithou.Th they could obtain o:,c',ysts in- the coca af tr

i;-tr'a'.'enouz and intram.nuscul rT adminiit ration of o.cyc,'lt ,

t.he:; fiil c.d to IccO.ver oc,-:. Lt fr'cnm ceca ;,'h.n :hiic.:.n3

r.erC inoculated intrtaFperit .oneall', or 'subiilc.,tneously. th:y

. sUr cd thatt c:.ocyat in:culated int'to thKe blood etlreafi tnii l.i

Lc r.enove: d fron tie circulati:,n by the liver alon'7 iith

other foreign bodies. Thcy rcpor.ted the pre'elinc of di;.-

inLCegrating oocyits in the li]ei'r of chicklens inoculated

intr a:-iu.-; ularly and Z;u-rez2tcd that the sp :r,:csts and

sr.-oriozoite mi,'ht reach the inte.tin-e and cca v'ia the bile

duct. ratn.,' : (19r. !) reported tihat Jilcn oor ,t s ist rl

placed in HillliFpore .chanbers grafted -l:ithin muscles, e::-

c.st.ation tooe: place inside th' chi'nber iith tllh help of

nri:yncs produced by the infiltrat ini Itul:.c'rcyte .. He al.:o

fc.,i1 engullfmint :.f pot'r':c oites by r;,acrophares in ide' t'he

chamber and postulated that tlhe miri.ht carry,' the cp:.i

to vr.ri.'io p:rts of the body. In all these: c.a.-e3 muci

lighter ihfectiio: occur-red after parent-ral inoculation

cori;ired.. ,ith th; infection.- occurring after o'r-.l inocuLil-

tion liorton--miith a-nd Long, 196I ,).


P:1"L,1ol,r '- C, C?.---l Co '- id r i.i ?

In'vol've, re-it of the C'--oa I :, .r th-v.- of ti'e aM:ll

intLc:tin,_ i on hiari.c.e ri.tuic oCf -. t- ali a in,'ction.

HOI,, '.'- 11, ;i the ceca -re ln'.r'ic-.ly rci-,ov-,-., or il 'v :ry

he-.,' inflection. the ter iu: l: portion of the la3i .. in-

t,:tine Oill be par.Mziti::ed. The lesions asaociatedi with









S t-. .ii iwnf-e:rioi in the c.:c h-:.: bc- n de.-cri o-i r'

': :-.i' '1'29), y_-_cr -i. i '. nul.d irr he, 1l' 1.

T1i, ,iia-tc.:i. ;: -i t of th.- .::.,cu Vn i prirar. ily InI.ol.;led ,.- -

i. : u rtial i da: :.a iz iue to th i e l r: 'c-er". of rela-

i'- iy 1.-r.: -. d -r :ner'at ri ': .LcTio;t pr-e -eiLt ir'z rhe

, :e.-rer ll'r.,i'a jl p i 01 t I -- niu i: ,.:-. Fr':'.ducti, :r ,'f

sr.-cific' to.ni hi; not yet tIe n d.o n'O trat-. al ti'u h'

pae'i'Leral di.nit at io0A of c::tr-icts of o'c -tz il lethal

to r. bt, r : .i. .t Io chi, I .:r-n i. r'r is, hl 9 The ? it ,.i-

I .-'1: o '-ct : t ire i 0t .- r' .':' il :ont aid 1'e I' S:

of 2nd h 2 .jcn 'nat i n i roe:r ::'i-. .i.nc.'r. e',,; anid loui hninr

of the c-ritir-oli l 1l1 iiAi of t,,: cCuiI, rnich:l- is: c time

-trippr-d .oiin to til- ba;: of the: siL.nuccs: arid i t1 replace-

r-ient by, 3 coi e CCO .ip c ofI nCI 1: i 1Otic t iSSu Li C': -.la..t

C.lood, d e:., r.. I. J .: .-:- 7. lo: p :nt ] : t ,:..:: c-f the

pa i'a it.- chief ,l ooc-,;st T'i; c.': i; at first a h.i n 'nt

to t .:-: :-.al wal -but aiter pay go-t deh- '' lied :nd lie free

iit iiri th-e l rre:. An i fjII't'. r-1, bir.i ...-.v' pa7 this cor' or-

a' bl'o clot' in the .r-O ria:7. NI't- ce:u-, .' rec.:.erinr-

bi y -i Ni it F r': : 'p-' r'aCnc-;e t r:- iri sl i:htil

t:iic:-'r:- In 11 l-hter iif'' cti cO rs r-:c cv.'ery ia oft- e co-,i-

pl1-et: rnid r:pli, .iut in i -i h v*i.i' i ,ecti'n .eco-.' er' i1

:ic.i an.l ti :- rhi.. : o. 'i't,: 't l. i- i ." i n ,coarpic tc: r- ::ir.. r ti o_. .

Tl:i co' t ini, O 1.u1 S h ',-I,:'.. l' e t 0a G I '. I; h to t th d y p.o;. t. -

':p: uL' rE ilts i' proi'c l n.:a iii,: ii of'r :- 1 1-1h.

cauLJe of ,l>l- h. ThI-. po ...1 !-iii :n.. nl i : nei .bra'e.s








betccI: pallid. Erytihroc'yte count: and herani;.o.o it deicroce

t c. hou t. 5I0 ':. of tih n:orinal on the 5th and ith d- y after

inrcc:ti'-n al itii 50,'- 00 E. t nelr ]- co; a,'c t_:, .ind the V'.slue:

retIurn to noiri-il in Toboun '3. day: iiqtt and lH'iPricl:, 1955).

[!-tt (1195) observ'cd lymrphocytopenia and h cteroI:hilia on

the 5t li day and 3n r':ai.ncphla1 on thie 10th day rollouing

infection uiL h F.. ren-ll:1. There iere no significant

chanc'ge2 in the raonoc;,.te and basophil numiiberi' duri -lg the

source of infection. A iarkcl'd lCucocytosi- began on the

7th day post-infection an,] p.er sistel throuLih the recovery

phase of the dl-seae. FPrat (1,0lO, 1941.) obLerved an

inlcrea5. in blood su ?.' dulin1 the acute rc.3aes of the

,di*-reasce .Cith a eci'e.rise. in th= rnui cl Clycoje '.'a:'-

1'741) also foun.ld a rise ii blood -.racl on the 5th day

post-infect ion~i ani a r'ic in blood cliloride" on thl oth

3nd ?thi dayc p:o t.-irifect ion wiith :aco;ipaii.,'flnj r action

in muccla. *hlo'id Houe e .e', P .er.,an i 1 '0) found no

il:pcrelycemia or chliian'g in hepatic glycogin, bu.t lie iiotjeJ

a. -cinific:unt; rc.iuction in the prla..cml lactate concentrra-

tioen on tlice t a;ni 2ni day: ,' after:. c:'poure :nd a i'ize in

r-.rdi.-e l:'ccocen on tihe 5'.ih r:'1 y p'T t:-irnf'ction. accordingjing

to D.-:L hert.y :':l i.' ric : 1 *" : ) during th -'cute sta:e2

of :h i infection, .:u..l: -nce rpro..,c'c .l1 in thr. cecu,.: r .ciuced

t'n-e abilityv of tir brain of tl.he chic!::-n .o utilize gluco e':-

but not Ih- :c-c di h'. rhI t:. T i cecl LO Cccid i o:

S c'.eCi'e ii C: -fere ice ';i th ro', .'Al .'-p.rp''crylC ti'-c I. Lo-

'dr..te utili It. io ': ay o.-cm'. C'-ni L.-y (1 "'I ) not ld an









incr,-as in adrenal a-cojbic cid lnd a r. I]a corti-

co. ,te rone coinc.--eitration: duirinic ti- _'cte honor rha. ic

ph?-e or the inf.: tion. Bertek .]) rI. ni clear-

ancO- or uric acIid in c*nick-ns inf ecte1d uth E. tEneill'

c:r:eatezt at 2 to 4 d.jys -after irfect: .on. Thii, tu

i. ecttcd th-t death i ri.t iue ro *?-c l I i ue .~- trl'. c-

tiro r .er se nor' io it enti'el diue to c'-ci bl.eling, but

to the f Iilur' to re.?over frorn an ifiitial choeI, resulting

fro:n thre d-evelorpm,-nt of large Inum ere of1 e i.do ienoi'U7 U .e3.

It is also reported( bv ,.Johnson:'r and eid i'197C' that in

-imi' c-. e'. the r-o s le isl in the ce in li .e birds

ill be -orze severe than tno ?se in d'.?ad bird ii iaje

anid Sunva 1970) tudiei the ,ritro. ,:n n:ta tbol im of

chic!:en inri ecte;d witl E. t-en ll.? anri noted that the total

pnId protein nitrogen decr i-a:e withliin th:- ct1eFOriies -f

all a:- on the 'rd a ni eI pec ially or th- .th day and

thii coiri ide. nit h there pF i:i 1f dev.elop.ent of eni orj:-no u

stages the pa ra.Zit Th~: as3ui. th?, t tr, e:- di. or-de r.

3re --Ocilated Wi t h many f:i tor., Ef ch di.or lerl: f1

ferment_-.tiOc' arnt. sui:.t io,0 0 -n inrilu nt e of Pnc et a b i, J. ri'o-

duct of th.? par it Tn cncli' ,rl .h:t c.n the b''a r of

iroteiD meti :3t .olir: 1 i thi liver f e. ?r.. :-.r i.h.. l", icF 1 .l hlOnFe-

occr in ,ourc hick.' ti-r.n iiin ?l:cni' brt"...: .

Lar'ly u :ori.- Lbv Lev ine :n1 H.-rI'e 1' :: 0, 11 Ci

slio:'ed thai1: tIhe ol t t.' : I r i : tel bird 'c:

iunmarle to do r or'e t ai i of .e 1,or Jo n-.e I:.y iL;Iz'l. : oif








n inf ect ed. hirric hcrin t iriulati d '.via the in :' ... : lo-v r,

Free,-,-. ( 197 0 i f n d1I that j h-ie thI 2 mus lec .air dir ,:..:tl '

t imuilatc,.t they a-re acl, to do: uorl: -ind speclaitedr thit

an itpp-irrlent of nervcCui. cosidiiction at the lneurlo-mLuculliAr

.i LrCtion :cciirr ii' cc' i i.ol .

A\ fc,' cc il] larl r'ezpcnse rPirce eft il. 1 1-?.2)

-sho'ed tl- t dur'in-; tlhe primalryl, in! ec ci n vithi E. t.:ii .: .1

hcaterophi pol.'moirpholnu.cl ar ,Ccella infi] trate inrt. the.

subm lc.C a it irr'e iri- n niijmb..:r especially ron tlihe- "th1 c,'

5th iaay: po's r-c:: pos;ur e chen t he 2'n6 gener .-:rat: c', -c Ii t C t

ire ,!cv:l.piran 'ii.l maturing. Leion -corihi,; has c:-.n

frt'qu:nutl],' uedr to compare quant it atLively,' thie rxterit of

,rroz- lesion.: aid pathaoloy'. Ihr ir:': ct I. (19' I 2 first

:1e c,:ribed a netl:d of .t c:ri r E t ri 1 :1 1:n :: on. usinr a

0 to 4+ !corin~i sy,' tiolr. T'his ccO'riv,.- r.:2ter. 1; i te- .n

ColloI, .el by ri.iny uoorker: (Ri pcorim ,lnd H-.- r .ici;:, 19I 5

Grd.iL-er and lP rr, 1954; CucI'ler, 193''; Lanko : 1 i ct :il.,

lo ',;; H r.-r-to'.-- nith et .2.1., 1961; L:.'n.h, 1961; Britron

*.t :- 1., 19.7; Turk ste? hen:, 1967; Dun::1,'y, I 1. A





I p'I: 3), P:iir and '.'i.:-ir ?'"5), L i'. -. l Ci 'P r 1, r' !'),

'Pletm:!y and Hi:.he:- 1.1'. ?-'I" ") H:,L,.'_.,-l.i~h.n eC 1.

,. 1i 1) used. a Zyst,' for r i. cr -,.cL c.npi' : r'A.in cfr l .. .1_.s

-:nd corrl 1 -tel rhi c. mi. i o.. :,pi .' i ._: f p:-' it] .1,

.eprn.i' I ; up i n the pr'.:-:,n? e o01" ?n. i',-riLi s t -'c i ol.n.Ln

and Rcid lC,0 ..,. i ._ r':-.in : .::,st;n for ;'Lros p .hlology










iLil el: .I.t''d .. .i ti for d..vel":I ii.nitJAl st:t ,:? in'

the : *. .:,:.1 t, t:r L... iO ." '* ri]" iz. tir,e *:Di-i rIi,' .

hoiul t -l. r ,ii-et-1 -IL'o i. i Ci' L. .h u 1 .F : r-i t ivC eint rli-

ti.- -ppe:. in t i. penuz rTL.:, ..L :t.-.- t ri r ,- ni.: r .: : pic ttu. ies

n'. b, ,TircJ tlP o decide .'i.thiCp leii;[1 2 iM. ar u: I t:y

,:,: i i .:a :. ;i.r'.::'.. rni. ''' i:O ( 0"17 ::: ine.l in-

r ti al v .1 J.' front ic *: s: A Aliniral.1ly dWInALywJ..

0 1, Suz p..:. -t '- "1. L : .0 *.:C i, .::.:.i- i ti ;zt l. ;-i.:-- l l'. a 0 In-

i !.Tri: ?:'" 1.'.-: 1 I z'.I. I "-:'[ tile ':~ ': i,) )ec i ric

.atfl lr:i. 1 man i fe t ti':L L C-o l-I i, H u, -I t h1: i it ii 1-





weeothu i: *1 ti. ctier e Cerat v 1, 1:1 int'itici-



-f tl1_ i i.i.. 'l:, '.c :i .1 nici *- .:4~ opi e *:r'ia-ii. latcri cf t.

i:te -t -in ?.l t iz-:iie f". t ': ,_n f irr, iiig p -,.th,, l,*:,;-:,' u'., t."', E .

t..:.rn- 1 1 i 'fere l icn.


S.-r flr n ni H. ':, n

in nui.t.:i a.rinim 'i iv'.' in iiti-te coat.-.t A"lth

rniu; t r'l C r riC!I: ite iji .:to.r i] n: tr: t ui: foiun.i

.i in t :hi irer' i -te i i r 'Ot.t:.t, ::n It'- [-I'I' Ial

t :' ; .i :T, o t L i h. I- .-.. '.":tr i '.e- i:- -l I. t .:i" i'. n)u .

Tii .. :1 : -.. :-L- 'iat ri hIi. 1.id, it nF h t Ca:- :. -": bio-

Ic ic vi.: :i.:. ',ip.. bet: .:-,; t'.: ':.-. ".1 -ri thc .m micrO-

:,r. .T i:rj ,:. ii. Lt 'r,-: h, .]Li" 1 :t L'- ,..:l-;. rn, iL Ve

* riir.;1: 1 i c C i i'u eiirY L';.cl c l-:-l c.: .x-: c ni y









their in._1 ,i oli -i :. cro-Clor:. nl I t'e r-e anir.ials ar! ..;'.n .ac

"c-..''.:"l'i .n ." :nin-is. In -i o: trast a "r i'rn-fr-':-" :iimrlr

iz on.: f 'rom iic.h it i not pF .:.si ole to e cover an' vi-ihle

orP -iiism. [a- i;: lLc'ator'Tis r"pl tects:- to detl'c t: bac-

teria, f i.--i, hel.ij rith pirn ..ite- PPLO, ardi certu-lin viruseL-

to cletermnine the ri r n-free sLtte of thi? anirim-1 Il C ljtocn,

196.).. The t-:rn "rELotoblote" Re'-iers nr -L 1., 199 ) ic

al.'o u.T.:i I r. -ferfin,; t1. the germ-f r.e anir.i'1 and ilso

niil-ns ,*:"'-yi n.'r .p'?i s ,-of o'r.anizm a. A "specific

p.-tllo;-cn-fr'-:-" ,.PrF) .ra ii.nl i on.r fr ee of Epeci.ifi d micr'o-

c' .ga.i:zms .nd [1.-,r...] te ::n;n to ,:-i-c .di .: l.Sabouri'y,

1'?5). The SF'F zminrail are fur.ctionally -nd struectiurall.y

ide-nt ical i.ith tih ir convention al ccuuntclrp'irts. hut the-ir

flcr..? .Crd '?.nu.,. ar:, to oone e::tent, cont'rolled. The

i'o'h i oif g- rn'-frnee or SPF a!niminal is a problem if the

part!.:, .' -.. r:al : -.ci.-: have > rtai.n co: ,ernital infec-

ticins .5 -1 -.- '1i ri lln.'-i in hicil:l n-.nd T:'-.':ner ni.'

in dog: R l.:-c t -1., l"6 ; Grie .;-.c.: t l., 196-, .- are

t''o inlfjt- tioi rI-,- iE : 1i .le- co--g, nit lly Careful s.elec-

tio"' ,:ind. i;c:.l3ati O of brc.- Iin- tockl free of tlie.e infec-

t lo .i .- ;. -i i 11 t ic"i o.f y -'un- niilI s o1"r li'- I':Ii

rpi.in: .:.i,' .:ci a eiut:l iO nfec ti O- -r c n.-r -.'*-. -. r t.o in.:ire

rb'~- r;-.- -f _.L: ,i L i 1 : L-rt Cn ti "' -.i. st; cc : i R e, :.c

e-t 1., "t. ). T1, .-e elamor- -Le p.ro.: d-.iUrecs e.-t hlis':

an ri .-.iai n-"r..c :. ri ip..lh-o -i-fre'e -nirkalE

fcr ro 1} .i C r?- ill] -A itr i n [.t.c lit ii-,?e sity aor

usin i: e. '; :I- :i. 'iirrn-l.- cf r.;no:-rn -ii:.-' ..e exp.p sure,









a,e tr',din11 anm r o t irport.nrit env i' -onil.-nt li b-,l:-.grou.) .nd_.

The po '-Sile i rinfluerice of trhe "nncro" .anl' "micro" en'v'irrn-

ment in *discr: e pr'oc.E --i O'n be FtI1di I<-l IutIEn .t ctoc.: c f

animals v derived froir. a br-.eed ing colon: .' i r' tised

iii--e r .' di f f r n t erLi.'i r-;iLri e- t:--c-onve t io liTli, 3FF aid

rcrm-tfree.

A inumb,_r of ror-:erp h'.'v attnted tc o rsi-e g-iem-

free animals itr'nc F-ateur' icpecul.a!'t iCn n 1n .-5 that the

hofr t-miicrcllor'r t elati n.shi p is lbl i, te But the in-

vert itat ions .)f' S,?notteliuz- ,1 .'') Cc.hendl; ( 101- ) ,

:Col'endy: and Uocilmnan (0! ) itCter (191 ), ClimEtedt

(1036., EB lzs n I,1T37a, l V3b) Reyr'n-i rs (l;iit, I.o'4 ,

1 6 )l, -GuS tafi con ( 1~ .3) nd Iliyakl:. (1 '' pro ,e

F-P teur ' --.ri 'inal az unri tion .. on 1:.L th. .;;erm-frE

anima.-l h- become a 'cry, useful tool for Atudy"in- true

hc'ieo:-LsaEi of the i v-atobiotic hol t, the. indi'.'ir al a:'t.

and interact t iins of iTiicroor arni smsin ? tnd tiie r. pon.r,-, oe the

ho It to theei c r -~:riani mri. Th'r. e li'ter.'izctions hi-ve not ,et

ocen il511 de'- ined althouc'h a fev- ,i c-ific'a,-t i _ic of llort-

hiost rc.l:'tion.-hip? lhiav jeen ecst':li ;h,-d.


The Gerr--Pr.. Chick.

Tine embryo of health.' tbird: : n 'it:iin-d in a

.,erm-free condition iiirhiir he h; 11 until Latchiing. Thi.:

has en,-,nbled germ-fre-: ic '., ti l nnd orl-r r I to

bt obtainel r'ith relative en .-:. 'er. -rre.r chicl:c-nz are

very popular as e.:perinental asrrirals nnr h-:'.' been








sEcce'ssE'ully used- to study such research pr!'oblem-. ac t he

origin of blood grr'ojp B agrlut iniri. (Sprin er t al.

1959), the gro.,th stimulation of diectary antibiotics

(Lev and Forbes, 1959), experiments on t'.umor'igencsis

(R.eyniers and Sacl'ste.der, 150'?! iev elopmcrt of parasitic

infections i.Brallcv t 1., 1967), and for a stud', of the

%tern-free state per .se comipasred with the conventional

3ninals ( eoyniers ct al., 19- 9, 19o0).

The gern-free chiclenn: o'e less clean than con-

ventional chici:s due to the high humidity in the unit

ann- also by the frequent occurrence of -ncl locall. a'3id

loo-s: nature of the fece.T of the crm-free :hilcl:kns. The

morpholo and function of thc giatrointetLi 3al tract

are altered in the absence of viable flora in the germ-

fr'e animals IReyniers, 1960). The intcstinal raucosa

hIil le-s lymphatic development anId ess coruiective t,

mas.- in germ-i-free chicken: and the general picture w:as

that there were e rore ablorptive elements but fewer an.i

l a well developed eletricits of defen3eG (Reynier et al.,

1060). Gordon (1960) fouln aboCr.t 3 tines greater nu',ib-rs

of reticulo-eniathelial cell: in tjue ni.coa r an1 the sub-

mucosa of the ileuirn of o.Jnr' conventio,-ni l chici;:ns th.in

in ,-er -free chici:en-. Thi i -ff-en '.:- ua lo noted

for "sc',ollen" or lobulele l'l:.cyt-s fnunl i within the

~tpith.-lii" :ci;d in tn.? rn -r 0 of r. o ll ind l.,'npho-

cytes in the Subnucoja i.ndI l1-mini nropria of the li.-;er









il utim. HCc tl pitli .:l i l c.:1 1 :' jI tLri t '::i ;:-r-t

in r:--rri-fre c;'iic :. Th: 2ni':.i,Lt '.f I:.ina prpr i i n the

;:t..:t l L st ctiudi':d 1.5 .. 0.''. in '- fre:- c hi .

anl. '.. 1.' i (.:ti' l 'hi ':c;: rE, I : n .::nd

L'D .:.c- r ,19 7-) re.F-rt that the Lei rit cf the Fs- ll

iu'testii .. I''' i'.-: t r i i .:, :.i tici tfhan i :. im-

fIe= *h 'c Enila rce ,- t f' t c ::'.ir, 11 : :. LI:

Chlli -ge in se..'cra.l 'p.-c2ies *,r ',lri|-fr., -'.i lriirrl- i(nclIdiing

the r'at, ui .:". e, rabbit a:.. 'i.i ,:3 r'it 'J,.:. t ,: l j ln -

iBr u:ic.itte -iat.rdol :, l''.9,. Ho.'ev.*-', :ec:il ,distent ,ici .1s

ni't bse ':rv ii : r -T' ree .: li?:e'n[ n d t uri eyI Lui.r''e ,

1>"'- 3 T .-: .2c.:c? .:-f t n 4; .ra- (r.'e C: li lns I.: I f .u d to

fe ci -nil' ':itl -hi.:rt.:-r in len-th tha, t ,:.-ce ch ci :.-ns

i t r r.'r.i:Al I -t r'ial li:ra I .: e 1 '-. ) Fr:.,

,r.:.: :b._r;, tion, th-: it r iint.stin, .ri -:ic-'ea f co n-

venlti a.l1 aid ,-'r r.lI e i nic :: i -e iril :r. H,:.- er ,

tb i ~r.l'ti -t lr it of tlai --e i 'L- e ti i i r, 100 ,i c e ri,:

tb:.: i.? ri .n: c', re te .r in cr ;.-. rf1 :-.:. L-:hc .'r'n cliicI:eIns.

Ti,: r ':c.s -,of rn-: 'rr'- and c.oa-vet i., l ?]hi-. : l- I I .i' ',:- 2l1

Si ilr.- ut t ,I C- r'-i c .? e e .: I i -ce t-- 1" 1 i r.l- t.

dtL cch The 1iyrrir -etic ::-:tc 1: .i : ri .L-: lor.. i-

ier i- t : a- nimi ? l .. ic. r- L :. -1 :.- --,1 -, sil of bitr--:

~ ''1 ind.:: *-f t .:. rri iy n *: 'l ":1l.-. vnt, i .-r 1 -1ii, a

,i-';t ii 'ferf:f nce y:e-,: foiuni:l iC I L Z .,.t 1., 1'-'.0o; ,Torri:ni ,

1',:.,') itr- ti en .*: rn-fr-re,- "-d cn, -,- t io i-.' ,:Ie '.cn:,- 'i'll.:

l:co--: c,-el i n.i-nc ..1: *:f : o-.nvr')' ti,: .'-,i1 d:hii.- ':fl,; 1:21 f[ jSrrer








size, full and more t.ur'id uhile il.eo-cec.l tonsils of

-er,--free birds i.cere flabby,, pale and inconspicuous. Thi

difference p-:rsist.-d till 5 :niinths of a;T.: nnd from th.en on

the diffcre.ncc br:caim less apparent. The rel. ive eight

of the trid.nt ait thr ileo-cec.l v?,l,:r ,iilcli c ont~ iin r both i

the 1e1o-cecal to1nils u Cnn cocistntly' andir .:ubstantially

-m..ll: r in :?-rn-rre cnic ;:ens I an in Conv,?,itional bin d-' .

The cornientr..tion of lymphocytrc. in the 1iiec-cecal tonsils

of the g-rmn-free bird-s was from 1,' to 1..20 of that found

in bird:; harboring li.ve bacteria.. Thorbhec:e (195Q?) found

no plasma .cells or s-econd-ary: nodule. ii' the ileo-cE ..:l-

colic junicticon of g-crui-frc chicd:: st 2, 4, 3, and 14

'i,.;e::. Thiy i 're3 founl in conventional chicks oC all age:

and in gerrm-free chick-: at 6 ueeh:s of g-,i. In ilhit.

liandotte Ban:-itn chiic.k the burz-3 of Fabi-icu; of c..n-

vent.ional chicks were larger p.'r 10" gri n: 'tody ueint Ulthan

that orf r-.rn-fr' ee chicks. In trcr-i.-fr-ee clhiic-;.r the splc-:n

:ias of smallerr rize (RE:,niers- et -i. 1 i9t0)J bit the. general

I.rucituree, color 3nd c on i enc,2 y of the Fc'r ip'.i';rl rnid cut

surf.*'Ces c ,r'e i.d-nt i,:al iiwith t!h'.t se in i'n covr',,-ttiron.l1

group.. Dis t.ril.'tion I f p ,l.aia cells ,:- -i il.r in horlo I

the group.: but t :is.. cell- 'Ic r? c- less often f, und in the

thy.i i., ilceo-eoc : jlun- t.ii;, --d f' l .lic:l of tihe bur.-n a of

tie g-crm-fr'e chick:s Thcrb :, 17 T -l .- outh of

cerm.-fr'e c.hii cl:ein re i rcd c.n .. c'iliZ.?i. p l if cld lic,'t

.13s found to, be ,omp'arnbl" a ith co tr'. l chi i s .e -r.--d on









i;itural ,C'c:,Trr i1. l ict i..ut th.: .Ar'c th r nAt r-c. t- : ..

t o b :. f-- r-r in : n-fr iI.- : F :ir .- P i:, I f.' ;

Forbes E t --1., 1'5'* R -'[icri c:; 'i. 1-60' fo: -In ,r'.:iuthn

?.ri1 '-r.roi. t i.. t : e I n rlr .1 bu.t e,;T r,'C i.tCt i. i-!ri

-hat:, hibilit, poor in e. n-free .:hili:' The redi c--ell

,,T.:rph _l,.j', ,', h'l r,,n-,19 lt, i i'i "-: n -lC i ll'..n ,l .: ',!:,..i t'.' I' ei t va ^lue:l ? li

th.:. rc c.: -I Ulcel :.ciIm: r'-c ic tic l ip ;i I -r i o andg

*,:,,V it|i: n:"J l c:hi.'l:cl E, ib.at 1,.n.:I': I.,- --: IIer-I'e t,: t i s:

Ji.r i i -:-tO i i' ,:r e : i nal *,'; : l:: e1 Ci r;ulsat i ;.I ly'mph, c-

m'e le re lsO hih enr..;Lth to : ta o. that i: thie r -o-n. -& of

l ivii- *ii '-,lorg ni 0? i a :r' t h'ir pr lu't:-t? n 1. i ef- oa~ t

On tlI numir -' of 1:.' pt'rr c.'tc I.Luel;-,', 1' _" ). Th',. lo'f

i' -l ':. i: L ': .' -rc t :,it'.-- c b i li fr-cr tio i: I eC :f

t h!i ,:!i-r r-':,;-ci' t- ji:i -s *:-f t ', g.r'r,-; fr.-e .niimn l (E li::a i ::C'.l

FPlillip-, 19.:o; T" rb, -.I' e ]. l':- Tii. g:.1 :1--1 ,

f mTm-- iarl .':ta rl-I.Lti: onr :' th ic t'l.,, in fr-- t i:,i or r thi

blc. :. *,f ;r--r -fr:c-, 'c:h i e l :o 1. l ';,:.sto tiinn, 1'i i .-c

Iind tnlii:-: ii ': .'.:!iti,:nal c 'i : io h 11 ii .- r-.um

F r:,.,j ;I.r? l:nl in riC uT- in : n-f r' .*.hi., .r: th: ;,

m? .1 r'-.: .i. I i; i !o n WI it LIe 10 i M in- lot-ul 1 .,O f

the gerwn-fr!'cc clh : 2 ''ir i ste'" :'r -......i .1 o' a ren-alt

:.f a t i i!'.o '- 'i t i i,: i :tit, i.,:. ; : 1 I 1 "r.7

: t _ _..1 ,. i -l, U -:. -, :. '. -' r.: r t:,:.l -

l j- r i :n;d t t ioi of n-r.:Lil i2 i I .1 c1 fr V-f ro-

ri '. L '"'. i .I-: r :. L- r: rt c i f ii :i ..r ni i- .-1. C'

tltr'- ti-.- ,:';'.erall .r''niujl :.,,';, _p.oi.t- i".

.,:.r'e p:, Iti'--.: ol- -i.ui-t'i.uir -'c tio,: t r .tcnt -il :.-i








cec- l]. con Ltnt--.I r, prs dli rectly related to t h. a, b .enc.. cf

, .ttr ii.tnt.-Zt nal micrc.'lor '. 5salish :nd Phillips 19661.

nid Srin i er i1 19":) found. the: o -:idsia ion- -re.uc tti on poter,-

t.-ial. of bLcteri-k-free c..cal content: of the cnick'en

strron*:.ly por-itive )while those of tlit coa-iventiotnal cick-:n

: tcr:- strc.'ily negative. EBlich and Phillip? ( 19u6) ie-

Fot-'L;cd t ithat th-i pH ir.s higher in all S.rmznrrtl : of thi: h. iu

in g.i-on-,free chick: 'hl.n compared to th It ae:n in conven-

tional chicl :. Tihe above stu.die. indicate thal. the gr rm-

frree chicl:z3 horu acceptable' nori.l tr 'i thi saind r productct ion.

In n-dtlition, parocii or c pont ineo.us tiunrs ar e not ct'mmon

in ge,'-fr'-.x chicl::. Th}.y survived ;:-irrai.i tio better

tL-3.an con'.' it i.n-il chic::; ihen the dosage ;;as beloi 00 r

at the rlte of i r per hour [c:L'au hl]in at '1., 1Pc ).

lio ,Tro. piysiolo.,ical -bn,',orn-llt; has been rep r.ted in

p et'U- -frte' chic:-ks. COne cc.:ilitio;n calle:;d ".jitt.e-'s" w-a:s rc-

po-rt,'d - Gor'lur t 1. ('19 9, i, ger'm-free chickL du- to

.:: ll l-.r Crolifer'_lt-ion in tlhe br'ain..

TIT: zer' l of cr-fr 'e lnimnls hin-: been shoc.n t-o have

Slou: c-lo'.'liiL c-.tent. an '..,' fw; .nti-L':teri aa;jl -

tilnil:-, Lt ..I. ,i r.il.e 'j it r.t- '. .teio e. : l tinii'i s are

pr, r-.' ''s to.:?tht-' ith -nucit of lI:ul-o.:vte andi

poor r aC oc t -po -11.- I r', .- : r.-,-fr'e :n ilc.1-

v' :.' .-,uscep lble Lo pr,.tho .-en-, -ithi.u. h, -iurpr' ingl',',

, ".-. r-e i 'er'' .-ur. -. r v .r:' Fr'. ct iv' in 1, '-

ing injc' Cl p- -t icl,-s ,r- de ." b;.:cI c: :'i ,(Lrcri;e:', 19'.3 ).

The "'-t I'ol ',' of .i n" iuf',--.-t .,., ,..;i..-c ha b .en -,zu iied

I ii ,-'. i I i i.' 1 .







P-,thc:.--n) cit i. of Int: -t in.l P:ri iet.? *: -,. F- -. 1 i-
in Crinotobi:ti.' Ho.:t::

S3 T',:ral i '-.- tii t tor- hae utii t : d it :' to t:.io t i

host: for tho e stiud"' :f c, rtairn azpFcct: of the hio. I:-p:isr:,ite

r-lat icnr3 hip. In contrast tc:, the r,:se arch on :'-:l"lic (in

'itr'o I cult i action of p.ii- itic or r nir sm:. o-hich i n:l ber',

di .-ct.ic tonir'di lr 'nia inr t ,M bi chremri :c l tr-' rd iriiTunoi- emic :'

charsc l:iicrtic: of thih or-sanri m, rn uce of zrcol.obiotIc

hlost: hIa. bien oriented t.r-:sr.i tlh in I-: '1' ctud. of thi

ei:lo .Aird pitho.'en-ris certain diz.1ase". A number of

tC.!.l:le on the: di-'. rlopf-i ent and .pith:-''Len-l:-si: of inte sf1 in?.l

pr:.t.:coa and iem inthllr.s of inman arid -nrli.Linil import ance have

t[-er done in ,no-to i otic: h rt s. i 'h :c rudi : h.a'.'c- lin

the re.liat ionr iip e.tr ,in tile n.c-t the parasite arnd the

host': inltestirn'l micr.-llorn ( 'chi.f' b1:ctcria nd fun,, -i1'.

FhillipE it Pi. (1 '95~ sho;.ed that Enranci "l- i' :I.clr i c

can produce pat holo'it.al lc .ionr in ruine.z piE-: onl:.' v '-

cr-cie: of E bacter it tuc :25 E.- 'fnr-icmi. (C':,i and A-ert

l:.?nr are pr' :ernt. Hoi -r -riothicr p:jr~ iitiC pr.l:' :'-

: ,oai, P.-r-trii h inmon- ('TricI,:inoi:- .1 hcmini: de'.elor.,d i 1

large niuLj .ers in perm-free- pnuinca pi--: hilll ip:, 19?.'i.

F nt:' rie ho ITr ch : .: '. t -1 -l- '.en zuboutiii-

:u l, irj iri :,:. i -nt -: ri-free ,-.ui-i pf prr.luced la ,g

l-:i 10 _, iit rimi ai' ?d, iin, ati:; 'n i! c n: :n.': l' 1 i.on-iil Luiri-A

plc-s re- ulted in di .'-pp.;ar-ince ,:f the r.oto-in"' in *a f'"'.

da ( I,"ton ,?t :1., .19 ).

E:.:pe'rmentail iinr.cti-i of ,.- ct 'i:.ioti' ~.,c-? ni lth

'l __ti-.o_ r. .:i_ dur) iu- an0d int i "- r -- : r - C 111. i








h:-ve be-n produced anid -Ltuldie.d (:a::';ton et a 19'9;

'cstcott 1971 1. In general nore- plra- it.e veI- lop-e d,

infescti i cn i trere ,f lone -r du. riion, iand m.Torlc helmintih eu'2

'cr I pr.cd'uc ed in tihe ccn';ent itionil thain in germn-free hc.te.:

CEO:i nophilia wa:. imalr::.d irn g'rm-f'rec mice following iena-

todlc infect in, but, no oosincrphilii, wer- seen in ccriv-etionrl

mice. iNoduile developmentt: ii t'h' intes' ina i l .iall .a: a en

in both types of ho.-it?, hojev-er the noduleF di.-app)ejred

fron, cc'nventionial ijhosts rapidly but .'?r -2lstei up to f.-

days in ger m-free m"ic?. ..'!eir tein e t 71. fl' ( '-.-2 reported

that t he larvae of !. ihi n .i'1 ill not de:ve lop to the in-

f'ctiE/. stl-ae in feces fro- gercr-free nice as they rn'rmallly

do in fece: of con.cvnticon-il mice. At le-i-.t in this c:" ,

th.i int-e tin.l micro' fl1r.- c rontr ibutol ed to the prlcin:Led

-urviival and es. production o-f the. h-Ieljintli rpec'ie.

Anrortnc: int.,reating s-tury by r[,uton ct nl. 195?,9) revealed

t hat the MiO nLr e lielmi ri in .i'-.' .arl H-'mn,=n .. r 12 n t '

which h drio not develop to r.nturity in Lhe conv',ntion'Al .l uin

pi.r, can do o': in the gerri-free animal.

.Jo;L'arcn et m ( I1' "': nd .R:-hov.' and 'Gric:rior

(19,67) found feline infectioua .Lt.:-rii ti in t'e- ,:rm-fre--

cit i s r lild, i:OnfAt.n1r:l di.:? .0-e ith s5" to : or, lc ':c.-

pen i t iyn lc it rop'. n.l :rlh .1 d..pl.lon, Ui. t .iitloI .t

noipholo-ic inte "tinal le.- ic'i ,: cliii c al Ci:'n In .2PF

c-.ts, clinic-l 1_-i.;, ultr'-- iructr .-. ter.L ion Oi' the

int.- tin- 1 n ico2.? and r luced en::y. ac a tivit icr.. Iote,-

(.JToi,sci -L 1., 19ti.7; Fo-l: and Ro1:Iovrl:y, 197ni..









I- iL' L- ica t -: In :i :-fr-', : tv urkr.i .cy Doll a it F r -iriti :er

Il'*-.. :'id Fr r- l:et :nd ['o11 1'-6.') i 1ii :.er.,1 ;hli t i: cL' c

fl' ':., i ;." 'if,-:t i, i':.oCC r e' ? cf i, nf,'. :ti- i h .t' n -.nii Ito.'

H. ,-' 11 i i ,-' ,' tile pPr :,t u' n l r,-i,--L: r-i.lIV. I. iLl, -

qui i'Ltlt EP'-dl- et al. l,1"- )I nd F,- :.l-:, -inr F i- I1 i r.i
l.. ;ie,-,,.:r- trt- ared n ,,, l c:'ti,:,l ,, inr'".- i'ving i prc'tz,' an .

o., 1r.1-- rt-i.i ') a i- 1- s ecies C' f ebt.rl ci; IL. c li,

r. r frin-i' -, or .. -,It i1 ) tf, inf ti' I't rc'-ep'-

titi ; l-uii :.,-W .r lin.z- r :t -.1. (1 ClI? rr:..crt:.1 that

thie bac tea ri l r:-q ei[ -meri t; for pr ic i ring inife ct i''i.i iz in1:cr -

I-.:pit,: it i i bacti ris-r iee CiicI er er d i 'i [' ie.t I r-rr,

tii 'cc for t i- diej i a c ii n ,onbac-.t.: ri-fro- e tu l::c,'. Tiiey

attribjt. i: t:- .:.-e role c- t.cter ia in ki.n. r1'.p ic -g n s3i or

ii-ife tio -' ei.ter-'iep:,titir tt r. .i:,- tihe ,.ec: p tir'.o Icn-

rc e t r i i: tiol for thi c iir- v 1l cf H. --n11 -ri iri -n.ce

hii rcm.:on:-. riav':-: bcen reo i. to rvi-e in b-.: te i.a-fr'e-

-. .\-. A enhancmien f viril cannot be ovc-'- r ilc.:i

tlii irnf: ; in :-. i7 i n- ti- ':'i" ]urf ,iin ir-c.i.c i:-. Ii his

S.,'l' :triie. c:nri tc: -.c -. -ba ter.: ri l:.: ti t i:,- ip i F ;illip

(l- ~.:-i -tat:rd tiat it a: Alimri t' ccrt iO n t]ha': tile nctcAri al

flc'r, r. 2c ;., t:, "p r' i'. ii ..-; :, -1i.1ti '...ie -.nv ir.:,- , r-m t, [,:.. ic:,l

ri.d h-lner] cal, for :- :.: .' t a cti:n a ,. bl i- i ii:L f liIn'Icn

inf-- t io unt il ir:H t1i i h t. t ;.. : m* on[ r tM ti: i E "

Ri:-c. entl y, Fhi llipz and Gori t i. I 1',:..I- ;--7 c .icmd -ion.trAte-d

ti: L lan:ous tic i r-i -I p,:,C i-2 alt --r t 'irulc, o:f f..

hii to:'-t -' rci in ar- : i:.,b -tr",--.tio 'or-:- cul. t-ifl r as








menaured b'' sutbsequcnt inoculation into animals. 'littncr

and Rosenb-ni0 1 1970) stu dying the role of bacteria in

mnodi f:.'in the virulence of F. histolytic found that th':

incrcased virul,:nce is associated only uith contact of

ameba withl live tlctoria and pcecullated transfer of anl

episonc-like v.irulence Factor from bacteria to the proto-

oans.

Reid aid Botero I1967) repo ted the Fgro'mth of the

coestdce, Raillietin-. c:cticillus, in bacteria-free chickens

and concluded tha-t nr contribution to the establit-hment of

the tapeuorn or intcrrerence from the normal bacterial

flora of the di-"stive tract occur-s. Johnson (1971) r.-

poitei the rvc':th and development of A w-ri~iia calli in

n ototbiotic chicken; -nd the data iiidicate an inhlbii l

of development of the. nienatode in the bacLteria-free

chicke',13.

On the other' hand, ?":lizh and P)iillip: (1966) re-

ported thrit oral challenge with C,.radid : 1bic.-s resulted

in crop infection in all bacteria-frec chicks but no in-

fect iiil ocuri'red in conventional chicl:;. Layton and

Sinl:ins (1971), in their .ztudi.,s nith *'"i-.** -r a-- ."" li-

sfenTricLutn, founi hisg.er nolrt ali tv '. r6 .. in ar-n-fri-e chicks

than in *sonv.ntio 'r- chic!:: (3 ). In .notori Ol ti :'.:1ne,

Lohl:r and Croso: '. 1'969, 1 1?l) 1 have described dlirrhc.-ngenic

-effects due to E at-ctablc" fil tr-L r. of 1-.oth cultur-es

and .whol cell v-3:ait.s o0 cl- r i-','liia coli and Ilever ut nl.

( 196( 1'.7, 1971) dcscrib-.d a polv,'eroaitis-like syndrome









die' to :. E. c l i n gc 'i-fr'ree pri.'z. 'T'h:- : .- i li- al l

inrdicat: that ncrrial ficrn has either a beneficial cr

i nita :roni t l,- ac tic n on .ni:i. of th-e .c patio r, c ie c an-

i-n:. AnJother i ;lt.ri -l ri n- role fotr the a~c -o iated mriicro-

flcl'o irn tlie liozt-prlri.l 1te relationship, perhaps, in

d. -te- r iriini n li..? t spe: i cf cit, I: the rI.e i tance cof th'

conriventionril gjine-. pig_ to Tr l yrnoz'-,ma cr-,. Hoiwever ,

a Tmnac: it., of the gE ri -r -e: .li ri-a p1i.:-' h1'ir. r'ed the

tr:.'rpna;omez in their blood f: llo u ing intrac.cal irnocul.a-

tin ( hiil ip: and H lfe, 1 ". In .con. tr-a t, tlhe

bacrteri al flori had no rol' int lt etab1 1 'li irent and

pat ho:-tn- i.::i7 o f E. ri'J- ietti it n the c1hic ,.'le intes stine

i HFe d:- eit nl., 1 ,'~ Ciar'l: et al. (l' :,2.1 l l' f oi.ua d ver-'

little diff r,.~ice in the pathocrcnici t o f E. tencill: in

cnvcr, tin .cial and ba ter i -fr'ee chickenj altIhoI'.i-ih there

I-s ieia. C.i' 1 to 1. hasuro in the appearance of the

2nd ,ene r.ationr mer:roi te iIn the f'e.c g-- rCnotrlAitic

hurt t In a rIL i.l're r-e2e n nt t ud", Vi.ce rid Buirn k1\ r'c..u,

auotFd bt:, Herrde 1t: al., 1'r:l e'.l: ported rio mortality in 41

gFOtoL.OtiC *'hii:n-s n i'fecte .ith r. T-.a1 1 a : c r.p.aied

to ,77: ri-!'rtalit',' in ini'crftc-J convention ,al chicl'en- The,

conclujdc.- that a ,clo: relay t i:rn:chi'p c :::ist: L :t .e,:n the

r:ost flor-a ?nd the i;irc.l:,:o n in Cir t cn- prelI'iAt l. ioni f *.:cC l

"o'?idic ic :ndroTe. -emp t i ,1. (1971) reported

diliaye- l .ic:-elcpm,:nit of C: do ,_eCnouL. 'tt-- of 2_. .'- n-: ili

ir- g-irm-frce clic i:., :cr-ci.all:.' 2.1 genr'-r:,tion c-iii -c.nt::,

gin, to:':tes and..- oocy,'ts. There :as ia clil:nin la: cf




39



ret iculo-FndiothOlial cells in the laniri n p i,' pri arid

.3.bnucosi and substanrti li-: lou numbers c.f i;onooinuclear

irfinT ann t.ory cells. Thus, the effect of the llact:rrial

flora on the pat.hogenicity of this specie. ri-nain

unresolved.














!C'Rl[:.L [rI'COBIAL FLC'ORA F C.i!Clii[


Th.-r imo'ort-nice of inteztinr-1 nicroflor.a to thli

weilf.r,? of the hoc t In e b-e, : rcc.-cI- e rl'. in thc

hiztc.rv 0. mi: ro'oiol,: .r. A,.-' :i reF. 1lt, the nr -.tir of tihe

int.stiinzl i,icrofilora 01 nT.n:.' ninial op:.:.ic i2 r e'll

d.:':uncntd ii the lit trc .Iturc cSniriu :'nd Crabt', 9ltl1;

Ui i rii. :le a ,nd Bri g 195':..; Dut'.- ?ni Sc? .:.il,:-r, 1'9.2;

Saith :nl ,ic.nes, 1,.-t .; S ,v-.va an 1 D,..b,:, '1 ,.;?; .::a.t: .a d

tic'ri rhit i'n ,'; S. e Ct n 1 u c. ?, l')"70; Ball a-t ,

17'i 1. In ;i'.i spec :' li.,e mirrofloir, ztuildiez h:ave

ueen li it cd t c t UT'-: ', rn- chi e 1:113.
Co:I: ; t A. i 19'') studied i-- effect: Oc aiti-

bi:otiC.: cn thle i"nt.a t in l iicr.:'flc'r of tur l::-.' po'u t

al Iqi er al. t 197T,':- 19t?,:, I tudied the irtestin.; l [ra micro-

flren of c.;mal health, tLuve;. s froL 1 d'Y, to : ,e!:h: of

rg and al:0 in trho: inf'IC tc1.. itl ".lJuecon ." The-se

Etudi -e: indicated I.- 't 'i turi :c- pouilt.; sho-t ''t-er

ha-.tching, the in t.-tin'i tmr.ct i ir-.' i'd L:,' -CV .i'al

-peci.s of r ,ct.-ri Thi i f-e :C.* 1. C th,:ln Piltiply

rapidi, r-e clhing- hi,:h nu; ars i I 11 A Ih: ir'.c -' to l .3

h oi~ rts of l1if Fin rin.c -'.? h?-v: z I .':, 1i'ii I r in other

i'ir, l ',':. (L. ai~~.~r -1,.- -,:, I'.t .. ", c'. 1 ,, t -.1

nith 1r.C L.) A number r of tieiri -is hivr stiille.d the








nornml baL ter i-l flora of conventional chicl:cn- (.Jchn-; :.n

pt .1., 19 ''8; Sha:piro and 5zarl' s, 194?0 Lev and Bri.,,

1956; L:v et al., 1957; Huhtanen and Penc3ck, 19c5; Smitlih,

1965:; Timnms, 1 ?.8; Barnes and Impey, 1 c6 1. Factoru such

a- age, alimrntsary Itict :tr*ucfure and function, diet,

fcedinE habits and environmental Cactore hav- teen shio.:n

to influence the bacterial flora of the normal gut

(Joihanmscon et ril., 19,4i.; :3rith, 1961; Smith, 19?65a, 1965b;

Smu.th in.-l Crabb, 1961). All the-e studies indicated that

the number: of bactcria of all groups: uere found to be

highest in their cec.l content: and progrcescively lower

nuImber.Z in the po-terior l-arge and anterior call inter-

tine, respectively'. The oirgani -ms constituting the maIjor

part of the flora ,,wre E. coli, entero.oc.ci (St2 rto:oc,.

fec li:-), L ctol:~,cil s -i ., B-Ct'.i.aii es Ep. and C. p r-

fri,-r- The ab:zence of R- terrnidrc cp. in the sm3ll

inte. ine, the preponderance of B- .rteroidis -p. and Lacto-

bt'-illus up. in the ceca, rand the loi levels of C. p r-

frin 'i'-nz in all Cites rei-re of panzFti.ular intere..t to the

investi-antojr.. Shapizo a.nd Sarle: 1.'-49' ) found the count

o2 aerobic and an'i-ro'lic L. Ctlori to t -imril- a in chic!:kens

of different -,gez. On the c'-n -.r','- H'-luhnnr,.n ain P isack1

l190-'5) found a prepo n-nder,..:c of -.;i,.-.c ol : after 2 :.eel:- of

age. Their re.c:uiltr .also in-iicat'd that in day-old unfed

chic:. t. flor. ccnisist.ed maill, of L. r:--Ilis. Tiese-

enteroco ,- ,si -gradua-Jly disapptearcd from the duodenunn after

6 d-avs of age. The cecui -ilzo showed 3n initially high








col.u'tt :.f eit'erococci ani a r3 robi;c bhctet i.a. Th,'ce wr,-r

replace-d by, ?rict-robic, typ-c at around 1i .i-ys of a,,; .

The normal bic t..rcial flora h a l3 crci rCepor-ed to

ini u.nc.- t[ l-:.st--cprecificity of corni par-a.i tes I.ile:to'. n

et :-1.., 1'5'?). Anitr cr int. .rc.t-rin phenom,-non is the

,*jcr. .a-' a-i, or i'iC'r., :--, :n the p:i pul::i.'.n- of o;ie r t-L'i; s

of ihe flora during certain patihc':logic.l coriditioinc.

Balii-h ind Phillip
inf:-.-'tio.n i:. tHi. cr :.p., the couni t of er.tcr i c.::,'ci in ii-

ciaz.,d. iJ?qi --t al. l '1?7C reported z i-,ificant differ-

r ce:2- ini t!he i te ,it-inal mi ic.r fl' r. in turl:c,'s iil.T,',l1a-td

uit an in.t-1 ctiou r:fiLt':'ri .i z. "blue,:n ") n;i ir and t n-

inf:ci :Tled control tur::,y:.. Trie 'chlicii ,wr- -narl ac Iterized

bLy i- ris- int t ota.l microbial coiut of thi- intir.? intez'

a ci.-nificant if n'lC ease iin numiibai f ,: colifo.rnic, li'. cto;ze

,i-:,nf rnienters .and cl :.-stridia. L -":: ,-t,1 cilluc sp. ,Ic:cre'ased

with severe' infectious cni'--eriti: but in.:r asked when the

dii.c. a.r vaz t- ild. i-licroflora c anti,: similar to. these

findinries hi'.u,: been ol.,,ervd,.:. y riith and Jonrr-o 1i ''3,

1 6.67 and CO ata and foriCshiti i "-i.' ? in pi -.- in.oc-.ilated

e:,:.. riihi e-'. li ,ith an enLeri: t n .' .-.i.a-:.-izon aind

S-rle: IM.1 'i .i iot l 1 that in t- .1-: in f ct a on, a Ct nM -

lation of t- I. '.irthi of C. r. -., c.,. :2ic'rrT d iitii con-

cu rcnt lecr c-.c .I I ,tf .t,--ci ]i'r op. ai,. 3 -ricc.: i c- blood

giucoc- level during thi-- tih t- 7 h -ii y po, I.-iii cf.i tioA .

Thic ;'-:;.' b: relat,-d to -n i t.rf'i.rn e in b, 1- .::. c.et- aboli-si-i




43


and a role for the flors in tlhe pa.'thogferie'Ti of cecal

coccidioc.i:. Thus, in cecal coccidioi., tne problem to

be. tudliCd is iihcLher or not the bacterial flora present

in tohe intertine aid or hinder the initiation or the

disose and rubseqlucnt deelopt evelo ofr pathological cha:ngEs.

A i.articulai'r s=pcies or a colabination cf species nay (or

may not) help excystation of oocyscs, subsequent liberatic1,

and ix;'viv71l cf sp.-.r'o-.oites and inrvacioni or cocal epithle-

lium, development of shi-ontu.: and,'1r gam ctccytes and

thereby contribute to the tissue .*anaage.














I.I'TF;RIALE AiD ihEi'H:DS


-'not tELctic: tba :ter'ia- I in'i- :nid FLC-!'reC )

c*hiCi':,n- ',:i' i r i : in 1fl.?.< .b le plastic M fil m i.,lit.or

fr.-.l.ii in. tI ie: netlc. of 5Br dl .-,' -t i ( '- ). All

1i ..l1' .t'. a cc:: Jti : :... :u.ppli : we e obtainr rt om

Lit -". m? -cur:e.1 To, meith.' : CmpFi:.',,i in :t-rilzatio.n,

riin:tirt-ncf e, cmid : 'er P ict o of g-iitoc'iotic ,n jiro i' .iiient

L hAm.ri"' -ir d i 'I :p t t. r.: i ili t io' o)f fee:id up-

,pl ,i .andi-i tie ,: h rji.: : for ,- rte riirin th.. mic_ o-

biolo'v,:~1--l -zrtuv i ;re ihe same as thosee de:cribbd bt

E'r ,'le:, st ii. (I1c<.i .

D '.-- l.9. or l''-dav,-old embr :ona ted ihite Le iOrn

chliCon '-g u re"'- :ic a a ed ron a comr.ierci li har: hei '-

fre-i ofii -Irr.-A'ii ajnd [,l .l.-'ma infection and incuba teI

at t e i-Aborato:r'. f ior to ,i-trodu:ct:ionl into the isolator

C:limber.- all :S ,r' : cnr.dlo at1 ltea lc t t': ice to inri u.-

th.- ;i bilit;,- cf t .e n t. r.:,' 'The .:ur'fac e of th.e :Cr

sh,-ll "s :c- : t rl. li -.--d b, inn.r:in; tR..: a D (pack-l il ?,

t-ibul::' 'i' n t: i 'for min ut '. i czolut ion cf


1:. F,. Su.'. Li.1 i.:.on, '1 ort ti utentin Roa,i ,


Flora' ci ,t. ate HaI-ltc'ic.-,.r'i Cain .ec..ii -, F or1.i 3








mercuric chloride held at 37' C. The eCgg uier'. then drain

into the prciseri 1ized isol-tor b:y nre'onz of an eZ'e cllhte

and placed in a plastic tray .containinil a cotton todl.

After removal of the Ogg chute and ceaiini of the entry

port, the entire isolator u;a placed in i room held at

370 C .nid 80-S5'; relative hl.ir.ildi ty for haLtching. After

h-tching, the eg- shells were removed anid the chicl-:ns

transferred to a plasti' urire-floored tasi':t 1 -ide the

isolator. Sterile feed and l -ter uere provided ad lib turn.


II. Froduc.tion of SPF ChiclIens

Chicl:ens hatching from fertile er _s obtainedl frcm

the same source as that from which eggs L;ere obiaiiid for

production of Enotobioti.' chCl.kc-Ls u;erE ini.d-iat.l;.' tran:-

ferred to noirficd Horsfall-BaueIr unit:. AltoC, thaer 10

siCch unit: urere kept in a room adijoining tios.: in which

the pla.l;ic film i:.olators were k:-pt for the prodlucti'

of g-notobiotic: chicl;ens. Air entering tlih Ho. ;fall-Bauer

units was st-.erilized by pa.sare throu;li sterilized fiber-

ilass filter media. 3tcril uat,:.r 'as csupplled in gallon-

cized bottles -tt-achedl to the inlet ti.iub of the.: unit and

the level controlled by r v-ity, flo;u. F,. : con -ist -td of

c.hickcn starter miash fre- of an' :u .ritibict Ics or .d-lcd

chemicals and iws of conpooii ior;j a; tin-. ltional Re:;'erch

Council (i;RC) standards. The fT.: u ... ateurized in a

hot air oven at 1.,':0 C for at l.: st 6j0 I.il-ut: .. and -isT,

supplied to: tnh chicl:,-.: s in a m tal celf-feeder inside









:.-icf unit. TeLup'tr tui.re i.,ns cont rolled elc. t ric:a ll; and

ventilition ': as C['.z-'rcci nw:-E tive prc:.3Iur-. Be-fcre

.:,i .-ftc.r e:clih u:- the utnitc weere cleanedJ nd -I-c:r-uti .d

uith hot detergent solution rnd stem st-rili eid. A.s r.'

Spo'; ible, the uiit.s Wer-e ,*p-neid :nly ) time during ani

r 'Fe rirntri- t--to cntc-r rie'.;li-h- tc liedJ chliick for e:-:po ure

of the c hi.:: to E. tenill3- ooc: stz, andi to renov.: deid

Mird'd Fel ioodically,1, M l inia.l labor2tor, tect: tere

c.ondul.rcte to cihec! : tie pI tlthogen-frP -? nr.ture or' the birds:.

All experiment. l :!tiicil:c, i rai ed in there unit; rc .?

fnor itlr.i'.d bt,' t-:ndis rd lu tr:, at(rry methods for thrL. follou-

ing e r.; ifi.i p: thoens:

1. tlE '? 3pe. -ic .: E ir.:.pri3 c oausin COc ,ldii i
ih :'aC i2 -!cei ;

the c.:oi-ion incest : ii Iieiiainttn: cf c:nic: en

(A-cari1 q,. H- ;ter -i_:s, ., r.cd Bailli triri ?

Spec i es ) ;

3. 1an-i nen llsi an F rst,-.iireii cpec-:ies; and

4.. rnel- -.,-rIli .


III. F i'Ct 'i C r-." 1-ii -- C-iy'k-ir-

II.on l"-n ..tc!1.-,d chic : ns w; ere tr.--i C i' red fror, the

incub:t,.r -d r-e r':'-d in L i ctric. 11 1 ,'-l- 't, it t er:' b.rod-

erc. lincter- 11 ii ed c:hi,:-lrri :rter h wiitri rio noiti-

biotice oP ?iddid .l'i,:.,-iil. r .i f :','irn- i C requir-m.L tz,

and u:-:ter er ma,]e- ?v:il..ble i. t ~,tqn. Before and

iftcr c:'-ci u.-,; t-hir pens i:.'re clean.:.d, ccri.bbed wiit hot

d' terI. '- t or lutic., :. Liu. - .ii ii d.








IV. Sour-.c of Ei:.-r _n t7ie Ul

The cz.ic :train of '.. I;-n--1 11 icolatcd frou a

natural sasa- of cecsl coccidiosi.-; 'as u:cd tnrougIhout

this ierics of c.::periments. COc;.'stEs uere produced

accordini- to need in .:iis:eae-free 3-;eei:-old conventional

chicken. Fecal nmtirial from donor chicken uis col-

lected at 7-0 days after inoculaL.icn %:it;h a tsublethal

lose of oocy:ts and tlhe fe-:l debri: and other gross

particles removed b .- :eviin tlhr:ugh 30 and 80 nesh

ciev'es. The .:oc,:y t vrc their, sodimlented ,'v centrifiga-

tion t.id allo'r d to srjortL1l Ite in 2"1 rotsium dichromate

solution at room te-iperatur'e and aftcr sporulation ;Cere

stored at lo C. Imc-dilately: before Lue in experimeTital

trials, all ooc-'sts ;ire curface-: terilized aith a '..5 .

solution of r-r-acet' i acid (Doll e l. l ?o13,. Steri it::

was tested ucinc standard bacteriolc-oicnl and mycological

proce'durec. Apprcr:.:i-n.itely 100,000 sporuiated oocy:,sts :ere

dmi iictered or ll for c:p r-.prinital infection of chici:ens,

usin t a asmill syrii .;:: : itli an at';tached c.:nrUlula to insure

depoziticon ito thli crop.


V. P-,t[-ln i, E- ,n--n- [ n .

Chici: ini c'iliatd 1.ltih oc.:';ts ujere I;cpt under

clo.c obj:ei".'v:tion. Blc i-'di or' ,ny their' clii.ical signs

vere nr-ted. E'lr,?l :-aiipl; s for i .l-c 1l cell volume' d.'ter-

Minltiot n u alid -.,rum n :0n.l"3Ic 1 e i:i:,i both before









Snc ul t -.tion .in t he 7tn ,:', after ,.-::r l.-'.re. LE a r,, rial

l.: ~ri'. mr,. on botl. th e contr. ol a d -:. :pr: r I ;ii

,;iic jl,1: .:-t n ,r pc '.-cc.rd.inii: to .he t.act.rii: ical3

rprc edr.n.re de crir-.e b':l : C hi lin.T .'.'i., aft-' :|:o-i.re

t. cc'.'zts and all th,:,; Zi. r ivir_,. cn th, 7,th dV:,- -ifter

.::p:.z.e L r.- nC 'rop' i:-il e:.:armined fi',r croF ?: lie ion of

ecal c :cidlio z i and thei ,Ec.il c. -::ni t it 7 e:,-ariiiid I

varI-: .i. dev. l p:. .T nir .?l ra3 ;, E.. t1 ci: 1 n: inf: ct in.'n

or':diL-:. in ea:'h in -oulated gronp war o.- pard .nd !vaded

for' macl i'cr :!:c : 1ic; :1- :. For hi -top:cath c'l:c l : :'-mlii --

ticOn, cr.il t iLsueI- oIin grr' o:' 1 1 :'i.n and c.Cial1

i ziz .- fr,:.m con ara' :le n ite. i'f :.'T cn'hicI:ir ,h''iii ino

'.:,." le ii:i' r'e fl;:.,'d in neutral ICi formna lin and

*:cti r,:.i it 5, or 10 .i- thicl:ness arid stain ed uith'

I i.-mlt'I'::'I -- '.:s in. Tne'r Y st emi i ar osc pic gra d1 in

i' l.ciis l d inro pi radini or rnr'.ic riir frasitism '*.uas

mcdelcd raft-.r H rt:. oni-S.,ith ,t al. '.1 9o ) az focllo .s;:

6 .-neie cf G-raling ofr Le'sione and Porarct it ism

E.-'t ei 'i' t1'. t re > pic 1 C"oo ,pic
L,:-. icn i, C ':dir, c, G riin. i'.
Frasi t ia i L.;.:icn3 Far i anti mI

Ilc d- t.-:' table fi. co--' clii'i t i :
leiE onS f:.Luid ,;7 1 : c.i r,:-
ful S,. rch

+ .3n-ll ui itr r .' Siill nir '.r of
Of le, ion --,i:. :: fl'l.lr.
by c.'.r. -i l t:arc
V or- le-. i


























t+++


['-i' oCr; c cr. ic
Gradinv: of
L,', sionr

lioder-ate numer' of01
1,-..ions uitn some
he-,or rn-'ti e


Hlum-rous lc. 1 ions
.;n. heor,-'orharge


l!ii.m' CIou lesions
i:ith severe ilinmor-
1h 'c rid C -3l1
enril rgerLut


-:t ; lcnt of
L- ion. an.-i
Par i i. ism

++


VI B-, ter c i lo -ic Proced riir.e:.

A. Drter -in tl Cion of" mcrc.,hil flora of tli- recum iL
c,-* -.-e i ti' CI -' 1 hIl -.' n

The development of cecal microbial floral in disease-

frc younct conveIenticna i ?hick.: and those ino.cuilated with

100,000 sportui.at ?d o.cyst.- was studied as foliowus. iiely-

hatched '-liit. Leg.horn :chicks werie raised on standard

electric-ally-heated battery Erocder- and f,-d unsterilized

feed ani inter -1 lib li. i .n,. Four uninfect -.: control anid 4

infected.l chickens o're n-croncie-d a.t varicLo s a-we intervals:

d=-<-old (control on l., .-.-. 1.-ld, 1, 2, 3, 4 -ind 5 weeks

.of age. Irnoci ul. ationi 'i h jo.-:'-, r i I .1jistedJ so that the

day of necropsy, i!o l.1. bte d-.i'.; afterr exposure. At

nc.-rropsv, the v'iscera "'r. e:...o;c.d ao.l the cecal pouches

to'c:ther witli inche: ol interior '!o nll initeLtine and


I;icroscopic
G'radling of
I'Iri s i ism

Inall rnuni-br cof 2nd
unei''at ionr .-chlizi-nts
-,d, 'or e-ametocytes in
Lcatteireri pro'pi. p uith
acomi asSociaited tissue
damage

flunerotls widely di s-
r i ruted -inetocytes
in localized fro.i uith
appreciable tirsuc
da ago

tlierli'Oi1 .schiszonts
and,' r Ranetocy ?es
iitl widespread d tiss3u?
d-anarg








p -.2t rior i.3r'e intent ine were tran Cirre to : terile

retr'i dish. Uhin:- aseptic pr'.:>e .irc., the .:e.al p:',hes

i:re opened., the e- le'i le tip I 0.Oj:. m i o, f an i I I jocul-.r -

Vn : ne--41 u .- insertel into th.- cZ:' el tcon;i: tz .'t ic.drIrin,

n1.. t ie rnjteri'i l ocn the ioor. .-tre-a!:;-d c.i tli: surzi'3 of

eacr:! : i. ti,: v ]riou .li ne1di iue .ied in a t. -' m- ri petri

dic-h or i o.:ul-lat'ed ijnto,- a tut c.:- c 'f br,':, Z:, r sc' 1' ii z lid n '.-l

In ca-e of *.Iry ,'c-cal intentent: or 3 f *-..rmei *.c- l C.,'re, -

rc p of te-rile pH ".0, O.0'.' ri pr 0Fhzp1n te i.ffer i.; used

to .'ofrt-n tie material Pri.:'i Cto inos rtion c- the -sanprilini

loop. A\-:-r t.ez e -numer tt :i: c.n Di 1, 1 bra iin h.:.r-t

inrfutirn i.'a r; ent- .'cr ?c in Difco R a ide-id-;:-.trcze broth

..upleiriente-.i i.ith 1i p.p.m. neth,','lene bli. ani 1.' .;'-ir;

an.i cocli forms on r cCctn.. ey -,Cr. TI' e me-id.iiu' uc.--:d for

,lete ir ininr tio t aia:rtobic populationc: ii 1- Dii :oR brain

h-art inruc ion qla- ar up iplem'nt-.'l i.itl 0.1 .' ., e tein,:-

I,',.'ir-o .loriie and 0.1'. bovine ru n. ,. -- r i --:- L.

enuner- 1 on Dif':: u .ulphitit pol:'T :-.:in ?ulp~i.l' i ini:- -a. -r.

For f n -.:i.1 icol-tion, Dif *:,:, ;.3 b:cur,'au d ri- .:.-'troze .,,-r n, t

Difcc R F~-,a-sno-Le:.'in e ar c, ith r i ilt a iot. i-c i-re i; e.l; ,or

pro, .to:- can i :olat ion, Lifo .- 1E-:l ,n -h '*ir:d .iu a: ..ei. Tim.

ci. 1 ub-t utl ion Mil tc 1 hcour " 2 f t *:'ol i* ''f-7i.' .. ; -io r cI'or

lc'E tr-.ii a rnl ;:'. ,-our. f,:,r all .. -t : or *,i ,'f .:r

in'cubL-t ion, th e -' :c-lon ie c r.:.n ca of the unin fec tCd

.cO t.iC'. L O i n t. , 1 i fct-Cc '. C c "' '? c .iirt-.':i '0_i*1 n'-.n

nu mbr b *:al: ilit' rd.


1Dirco Lab-or.-t.ori s DE -tr.-,i. ;i.- .T n "''C...









B. D. t lrm'nr tin r of mi.'rob inl flor'T oi the cecum
in '.c -i i--,re'. -, SPF. -d c 'pI 'nt i c nIl chi ck- lns

At neertop;y, the primary isolation nedia used for

the recovery and ident:ii icat ion of bacteria, fungi and

PFLO from the ceca of the germ-fre.., SPF ind cronvention.-l

chicken.; are listed in Table I. The criteria listed by

Breed et A_1. (1957.1 erze cnplo'ed for characteri:zi-tion,

idenrtific-,tionL, and v.'erification of ba teri.al species.

Anacrol ic plates w.ere ilncutated in a G0sp-cch:RI A.iiaer!,lic

Jar r:6'0410 usin,7 Gaspack g1as generator envelopes for

generating:, h!dr'ogen and carbon dioxide sac. Mclthylene

blu' wia:s uI.ed as the indicator.


VII. Fac-:ed Cell Volume

H:1-,tocrit values Irer determined by drawing the

blood into a heparinized mic.ro.?pillary tube and centrii-

fuging the blood in an Irtrrn.t ional Ilicrocapillary

Cent-ifuie" for 5 minutes at 15,:''00 r.p.m. The packed

cell volume (PCV) was directly read usin? an Internati. ial

Capillr 1 :: Reader.-


VIII. Tr,,:-.1 -<-'r- PFr tein

Total serum protceiin ,as dcEtert'il-rel by tihe method of

./eich I1b.:-um (1''46) 1ith Lclii ht. mo-lific: ticn: A standard


1Baltimore Biolo-rical Ln'bor.tory, Bal timore,
[i-r,'l and.

-Inter.inati nal E.qipTi ent Compa-.ny, !eedmamn Heights,
rI; .-a i c r hu .- ts.











T .Yhbi I

Prim, ry IT.-.u,1 I. n ,.-.=i U.-e4i Cr tK Rec ler',
.nr, i ,' ntifi. t-. i i:' n ,o,. Ev .ter, i. -n F.i' F.,, .;i fl ro,,
idecr f -c 't i.Li t'ntori.:tI i i .' Fu' .i Crl: r
C ii C i' ?,i.i .' 1r-. ic -] l ii,-'I:c 1




Di fcc' .: id--.tie: 'o e broth lth 10 p. F.m 1.rnith:, .ne il e
,nd 1. ;, ?., -

D l cr.: lo zi':-' _-' ir Ait -. i ini. d btovi e bv i loc.od

Dire." b -in hi iart i','fu:"i--r' ._I'-- 1i t7l- 0,. C '.'. c.' :t ine--Hcl
aind ..1 tbo ir Ierurn

Di f'.'1 ?, i'ri 'i .van.'i: ; '-cuic -gr- r

DiCfcF, bl,.1 -.? id r.-- uii t 0. b Wooo Wnc.. a' r and na.m.-cin

Dif co -, lum bi. t.ra Ot r
D i f.c-R 1 t i 1 c 7 t 1 *!

Dif,.,-' enterloc::.ci pr'-,' .Irt i.'' bro '-n

Difcc."o ? Hic,.cn> ev.' _,car

D1f- h.:.W e Pa.-:n -Le'ine b:-.-e lir tr-iph,..' 1 t tr:. olium
chic'.oride id ntibiot il,-

Ditc..oR PLC: t:r t. ult antibiotl.:.-

D'Ifc: s'ai:..:iu u.id de'.t' i 3 :C' i unti .iotic-:

Dirc,, s. iprn.t.-, 1eol "r.,;R::in 1 -].i l-. i: '. r

Dif....:, liioni-ir il' a-.^- hii l- 1-] '- ", -i :,r

D ,irc-," tI ,iii ;,lc ollat-: rnC.,iun Uiltilio t *i::-.tt --, :

Di -co : ,. 80, O .'i:le,: :" '-' : :.-: 1:J

DifC.r t ,; pt ic-: -; ,-- ,' c.y rcth








curve :as plotted uring v.-.rious dilutions :o a 10a bovine

serum albunrin solution and cor.respc.ndi r'L: optical densiity

in a Turrnr Spectrophotomnctcr at a nave length or 540 nnl.

In the procedure, 0.1 ml of url:nouwn serun and 8.0 ml of

stable biuret reazcenrt2 were mi:ed and incubated at 370 C

for 45 minutes. The optical dcrinity tias then read at

510 rnn. The total seCrum protein wa11 directly c:-lculated

from the -ta,'idari curve anJ expressed as grams per 100 ml

of seorum.


IX. S run El ect I'o I lre i

ElectrophoreEis of .;erum proteins uns done by

llicrcazoncr Electroph.horcii3 on cellulore-acetate menbrarnes

usin, pH .67, 0.075 ionic strength barbital buffer. Using

the applicator, 0.50 )il of the Zerum -ample was applied

onto the membrane -nd electrophorcsi:ed it 300 volts for

35 minutes. Tre membr.ric then was Stainerl in Ponceau-5

fixative dye solution for minutes and cleared in 33.

cvcloh-.:anone in absolute alcohol for 1 minute. After

dry.ing, thei mEembri'rnc was canried in a Ilicrozone R Denr it.-

meter and tie ch-:'-t tracing: evaluated to obtain the

co..ponent p 'rceintrtgs..


1G. K. Turi-ncr ;.T ai..itest Falo Alto, California.

2Hycel, Inc., Houston, Te::s.

35c--,i-ana I:n t run intc, Inc., Fu]llvrt on, C-~liforni3.
I1Ii









':. E-'.r',,i, re' 1.0 BE ,Ic',r .i ,I Fi, n 'I i

iniig.e ?p ic ie cr c.-tjiilat I o o bc a .::t riia or f'.rl-i

o b:' u ;i -lon iln t'cr l l i 'c f r r rr..: rI-t' : 1 ':<.po

l'ere -ele'?t-:'l *i th l:; l: of iicroi i il iC:i :ltt fromru 'L

o01 c:Clnl'. tio ,inal clici:ens. i:'.ii-i'. ehri 'V sev' C. :2Ci..iO10si2.

AIr.o.irmratel: I .'. ml 01. to :.:-.ir broth' j cul t .I of

tie rErpective IrnliTin 1 A1 dmii st-rei oral -. u-ir.t-i a-

cL Ifili LI Iii *a:- 'f f. i c li: r th'. n i' r p. 0 5 mil

.:, h ..?.' :pr' :u pr.enIi,,-o un :" adniistOred. '. _.] i',ic,:!r'

W s Lr 't in? ife:. S t:iiuriudl de::tr --c: tI roth at .0 frc

4L hCLirs anT-1 l, 0.5 r of f-ne ro-li si a":r d ini ter-:.'i .ora / .

E' cI..; ill specie 's er.: dniii:t red lot.J.- before e E.

t,';- ,l ..3tc t ,te *i":i,; f'i.-1.:1: o c. n i'i 35 ;. rt'- iven

, Lr::''L r? prior to0 c'oc'st a:dini ic tr'ati':.n.














RESULTS


Dc'. loo-rint of 1 ie'o. j -il FlPr-r in CccI c r.mi .-,f C.n'.r t ri i oil
Chic:e. n Inocul.atcd ith E. tenrellr and Uniniculnted
Ccntrc'lc

The predominant aerobic. -.nl ans-EicObic bacteti'ial

specilec in the cccun -.t different ages or control chicl:s

and of tho 'se exposed to E. ten,'ll. oc:,,stz are listed in

Table II ani Table III.

In day-old ind 2-day-old conventional chicl::, cecal

organisms i. ,-re predor.mnantly enterococci (3. frc':i :1. 'ith

E. coli mal!:ing. up most of the remainder. In nioni'fect-d

chicl at 7 days of age, enterococci 'Jierc- not round to be

the preIdominlnt org-nisnm in the ccc:, these l.ein r..

by F. coli and Lactob-pm.i ll, -Zp. In i. t,-ne] -.--infected

chickens, the enterococci ,;irte c-restly outtnumlbcred Iy

other sp-cieZ, especially E. c,-l]i eaid E- tero:idJ -p., but

InIu bears of L-'r.tc,',a ll'.-' 3p. erer greatly reduced as con-

parer- to noninected controls.

On the 14th day of -'e in nonnini-ctc:d ccnv'entic nal

clilc,:s, E. cr.li :a ] [-. :tb- l11 ,,. -r. trre p-'edorinnnt

'with f u w enterc, ccci, .-nd i ':-t -- il s p. 0r'e ri 'e.. In

the infected c hicl:-, F. ;oli 1l-.: the rist predcorninant

csp-cic. '-nd L-'ctt il 1 -. sp. .-re not detected.








A '1 l ?.e da ysz of -1e, E. n. 1- i :,ind Lm'tb.c % ill

sp. predor-cil ted, in the no-ninfected nr,,ir, jurile inr tl

infected prouip, 1. ,:,11 b ,:t.r.i.e: sp., enteroc occi aini

L'::t"h- ,ill .i e dt*etI ct-d,. A ctimulatition .:. Frro.th

of E. e-c-i, FPcteroide s F. nid 1. fel:1lic !: s o.:berved

during. E. to.nil l- inf:-ct ion 1hile the ro:itl of L i: t,-

r-'., L F Lp. .s c-upprezzed. At 35 days of aie, the came

pattern rwas obb:,-rvcd e::-eFp t hat in chi.leen: infri.,ted ilth

E. re :l ll Ql pr': riri rT~:7r. and a!iIaer'obi': fere'l s:trrepr.oc ci

tere a. 3. detected.

Fui;-al isolate: 'were rare. In c.-d,-ol 1-cl non-

inf'c: ted criiCI:0:n C::.nlid-i 'Anrd Illh or specie: were detFe ted,

but in tl-i inrfec.ted -e.: cril;" iiao r pF Cies3 c-re s ien.

At 1, 3 a,-il 5 uwe -.: of Crndidj :peci-:'i wI re ,d tct te,

in nonin!,'ated g. rolp'iFs and itn '-, .:ek!-old infected c*hi.:lenr

m:,r c ,:ies wre detected. In 4-weekl-old .hi ?f:en?, no

funrl i ijr-e detected in eith-er roni nf-ecte.l -itd inl'ec'ted

group.


EB-?t ri, l I :l t.--: in ;-ek-0 ,l SFF -m.l Cro.rvcnti.r-n-1
Chici ..i In'c.:u-l] t-. itih tt A !.- L'.- I ir-ic:i..:-,l:tc.
Contr.'.1

B,-Ecterial izol-ates in '-teak-c.ld SfF .ind "o.ivent .nAl

.lhi.Il:cnr iric.u:i Vted wlth E.. t-. j.1 ,^'r'e r,i,.t'.= or' less

sir il ar and are shon-r in Th L : VIII :nd I.-:. C. nor-

r i-, it:is ic..l lted 'ron:. infcc:t:.J -nd noninfect.ed SFF

, ich!i.lci 'I fl I'Cr infected conv.i nt ional. c hic-kenrsE m otrc fre-

quCnitly tlhan-- froma noanin r,. -te. conv eini: onnl chi c-ken:- A









stirIulation ..,f t grcir .th of C. r-e frin'cr,-i" Iwr nol.td both

in infected SPF and conv'irntional chicken; (Title X) In

noninr'ecte:l coinv'-ntio rnil chii clens, C. rerfi'rin~-- n t'.

icolatedi in only, 2 trialr while in infcctcd chickens thick

spa.ci-2 wo i.solited in ill the trials. The number of

colonic of C. i.,i''rCi.n:--;nz Jvecloping from a standard

inoculum was hciicr for SPF chickens than for convenitinal

chickci:. :cc iu'rence of funi;al ;p--cies was Zlso fre'eiqucnt

in SPF chiclc-nn, e-sp'-cirilly in the noninfcicct.d ones.

Carid -i s pecie. I er. isolrate from nonii'cct-.d cont rol in

4 of the S trial, uhile Co-if'l:ida specie. ,orei- icolatdied

fron ,cnly 1 of the 8 trial: in conventional chnickr ken

ITables VIII and IX) ',' species r'-re in'frequent ind

i, re isolated only. in 1 tri.il fz'ron a rioninifectcd colivcin-

tioi-ial chlckr-i i-c,,OLilaited with E. coll C. n r''ri'i -.-ci,

and S. fecilic..

IFathclogTy due to f. toanell in Lacte.ria-, fiunii-

and PPLD-free chickens i; rt'ported in Tsble I7!. Clinical

s.mopt ora like bleerlding, aiiore:: uo l: ie, c l: i. drooping

e'or'e not nrot --d in i ny, of hL. ese chlc r.:-. i;o lior:it lity

occurred in 32 of t he:he chicl:er .- raiz.,.i -t teri :-, f'u,.i.i-

=-i FPLO-frer. The fimacru.opic : rdiin of :1- ions iat

ne.:'op y, in all theSe cai6e ti..-.L riCc t i'. ct 3incv thiCre '.J.a

no visible tthickc.' ninr i, he-iorril? cor'' fori-tion, Ci"

slouii-iin of ri'- f muce C-:.l -' n !rT.-- cnt n"r !L not

noted. Tlis appe.r rancc of ceca, li'vrr, I:idn:y-y small and

large inte tinll, burE-s of .'abri ciclc, ;plc:-n, i '-rt, ni.iuclic ,








ir.nd l1.uL ver'e n.orcial -i comTpvred to tie vi: '? Ir'

non iTi'.:'r;-,d controls r.-ii-e. 1 Iccteri- -, i-.r.r":i-, "FLC-Free

andi a31o tnm ce rai:1 i .-ec i i f pFr tho-, li-fr-';r and c on-

-nt i -,ll: -. Hi t.pathol_:' i l :,-, tic r.: u a; ni- ti2 l wu

d?'m-1~ ir licrTmo'rr'h '-X" loii,'kiri c- or tkhii',! i:e ir"if';;" of, th..:- ijt ,-.a3

e'.'i- nt .'-.i ni i r' o' o.pic e:r.- rniin'l'i' l v.e e -. I-1

sp:Fe.-c.r d -c -, r vr -ve ind .',iid r' :-.'', .a":- i v''lpFri_ rii since e

endinseinu T ~sl. ere :te.i in trh, ,'pi thr 1 i. cell11? or

tbr mE rj. ;Z a, :p. oec,.:--.11, C ..f the 3lani funii TIhe e e-nd.i 'e-

nous z t u tz ;-': ivere ident i _-l '. irrn-auri hi n.ts and

e arl v 1 .-ar:- tc ,t : Il1' lr '' e 2 n-i eC C r t i ri n l O lC CitZ

containing. mtiiure mero:imc: uwere son. C.l -occid)iosi

,- c .-cr-iibed t' T :T-cr 1-.' ;a.rid T,':::r. l. ', '.? 2 i i,:i-

ni:tc -: in r- e-.c ie : .C ,'-:en' a hcrrn :.p. ,-c. c: : **.-ment a]i t:.

: .jid r.fll 1 iiC.;0ilin cf C C I' l -] c:'oc'tc.

Ti-er.-e .LJ : no decr'e :a in ie tci' :ric t V'r ]u'-2 in

L.actez -, frungi- and r-PLC'- reer c, i. t:.: e:t..,. ed t. -. .

t vr.-- 19 L- 'Ta le .*:I) On the contr i', r hcrit crr i t val uc.

incrr e aza-* from a m,en prt:-infe.':.t i.: ol:.i-ne otf 27 to '. .n

th'- 7th d.ay pr st-infectioii.



It th 19. *e l1% C ,..is.e.d ri _l i 1 .n r ," -" t ... -r'. n it C 'no,

pro''round bl.di.j n cri- :i u ';i T hie ) ii toIlo' i. al ,',

the t izs ue .:ar.age :as. n:rli i,-ibi- b,,; e-i.do-:eno- s itn _E -
erer e:cn, e p,,c-'. all;,' in.-".tii : j i ::.. :-. D i -.d ti n of

nuiC.o '" .s. rfit m ail. H-tmmt it vili. ." -0ho,.-.j -n ifnc"-rea3 e

of 1I'. in tiie-? chic cenk on tlh-: "tjh 'l po -infection.









In chliil:cns h1a-rborin f i' or cp?cier rioder-ate-

numbe.'rs of lezions i.it I .30-ie heriorrha ;C Ind thu: ike in-

lias noted niacr'o.copiC"Ily Hit.'tlc ical..', t ere as

moderate tissue il'anage and l'ir- nunblrs of endjgenoiu

stages including oocyts u\-re seen.

In chicleen. mnecric'onta tinated.l ith anaerobi fec' l

stLreptc.occci 'nd :-pC::eDocd to t, '11 there were no

vi ible le.ion.? r hc .:r.!i hage, bu: hiitclo ically ndc.ge-

noCu st i;.g s er,.I evi .ic-nt. Ti. ..z.: iiar.iare, -.lougI thing cf

rnmuco'sa, henlorrhalo, :arid lar'' 2nd .arne'iat ion 3,.CniCont

''ere also abi:.nt. He.-- tocrit ,values increased from a prc-

ce:poz-ur' volur-e cf 17".0 te a p':p't-c-:posuire volume of 71.5.

In m.;'lic Fne-is mi)jnoD nta-jnrt .ted .ith ana rerobic. fecal stli'r',n

cocci :nd a .3i.tpen.ion of killed C. oli 1 Cr-''rrinreur.

S. fec-alis, infection with tir. ti-n l1 did 1not produce any

clinical sy'mrptorm, dentl, vi1i1 ble le ions, Ihemorrhage or

thi cl:enin of c-c l rnuc,'z.a. Hirto logic illy, ino tisEsue

dr-nage-, large 2nd gcn rnatic-i cchlc cnts, or bleeding '. a

:limona. t rat ed, ibut i:Linatire sc hizonr- :'r- eoe .e in the

.plithlic li- cells of tc nucc~.'a. T'h1. nr,-.to.l-it v. lues u 're

-7 at JnVy of 1 .I.c,:ulatioi '. e l "1 .n the 7I.h .: y post-infect i.n.

In chic':en n-1 :c.-:c n1. . i -1 i there' 'id'-.-

.:p., .-., r'**ri.c --? E,. l L ,ac o I:. c,. fo,.;'O '.". liklc A.

'* ro n-';- i .;, mod rate ni'IL'' 'l ofr ie ;i : '.'ii 'herorrhae werre

notice. '. 'Tl-1le VI). D.i' ,th .u- t.'. 1 c :.c i i..:i. occurred

oinly1 in c ut or 12' .1} i cl::n.' ; n. c io t.n.;ii ',td rl ith 2. .

c',: li s, and nIo ior i:.l it', c...iurred in L;heroc ion..-.cont i n tcd








j ilth >,thl-'r .p':ei' e. bi C a, I-cte' i In CiihiC. t.';,t, n, ;!i ccnt;i_ i-

nated ,'it-h ,"li'- th: r-: i'i. Ra t.-:td cic:' ':r tI 1- '.d

tP: cc. i'i. l te ii:s rn pidl F at r i all c c t l a t. ed bl,:o in

lh,3 c-:c,: i/a. chlrU .ct PriSt .c.llv pres.nit it Ph.ln;,- ,'ic,1c:eni .

H'-]:atc: cr'it v.l i -:. also ;:hc'~l 1 c.' .,jt.iir:.n iii *hickens-

io.ncc.t ru-tir atc i uith bacteri a -i.. iin cted. iIl E. ten'll :i t

,'-a"bj c :I ) Ci:Tm arat, lr a C ic. I, .e a C i ,oCnoc ,.il '~T i rna;te-

-it.ler .il'i F-rrtl r ie.-l l p., : lCri s C, ne, c r i .:

iev:lopei ro er.ti e t:o ?c r: p t h ; .l i hei n -lp.:,:, 1 t .
i-enrti- Chicket- wt b,,. : n'rtl.t 1n teL ilth F. *rl I ":r L-t, t,-

rcilluit. :r.. .iv:-lcrli cl.' tild tP rocd rata lvajcisr, n nid

tPi-,::- IC" rlc.:.,.'tami',i at*'_l: -i i. .tti a-rir'r ,.:bic f-c: Il ttrp---r.:.ec CC: i

Pchot-..e littlJ i. .-,t ol,?':,' a.c:-.ert e: P C: :. I. t .: p ] 1 i-.

Chic::tc irfectiid .ilth E. t.:i-- 1 a pc.y:c it.Irri-

nated .t b :.r mc,: re Zpacie.. :,I biact..r'ia c:r funhi; zhc:.'..';,

le ;'i, -, int er d i 7.- atf- be lt ire.: -i t hi. e : mCrn,. c.,:,:.i ta tt i ted 'and

PFP r :, e:n,'enti.r-l chicl.e s i. Table 'II). H-a-;,y r rt:1 '

- a' and t;pi.Cal ;ricra c, plC nC icr>,c.; ic 1 l z iC h.-

ic'ted in chl ic:-etc prel 1;c i aii id th C. r. -rfri, :,.t' a-i, d

-. fec- r11i Deatl ,i, typical l ic:, i de'-v lo : ic c :-.:.n ii.;ro i

d-r_,-c.l.1 chii.ien. pol:.c,:! anint -a ed ith -,ti ',i C =jJ .o p.,

'-. r: 'fri '' :- r'.li and .. .: 1 r.'- .: :'r ed 1.0

.. ,- r'e 1l r -,ra r -t. i v'- ]:,', palh: ,l,:. c:,I natl iF' s t- iC-n-,

'..'ere mo er- ::v re' in chici,:,;.i p.r l;'o rt-t: n:.- ut.d it :' iletr il h

I. ---ID .... - a l -1 .. I I -1 ,. r _--" .' :,- i :L : -'id, t.

. -: 1. thin ini tAr.hocs p lCc.it nta' ti r- r i ,ri- :

anrid C. ccl .i, L cti ci1 s c F' '1 -1 li .A : .ci*t.i.c









of b-ctcrin -:nd fnzmi fa'.'ored dev.: lopment of moderate

l C-ioi L-., but,'- lil not frvor development of Ereater p'thol-

o':,;. Ho'.cve.r, the p-ihology w3a morre s evCere than that

ec eit i-?r withi tnti bacterial :.pecie alone or fiunigu

*pocsie- alone.

Palholo -,ical miriife:totiions in S.. tcnclla-infe,?ted

SfF chic:'ens Wr-:-e typical of cecal oc.idiosis Table VIII).

Clinical .ynptoms like bleeding, anio'r::ia, droopiness and

mortality,' cri noted. Thirty-thrce of the total SJ

chick:en.- inocillsted lied, establishing a mortality rate

of 3 ilacrc.scopic as uiell as rici'roscopic legions were

otserv.d in all infected cnil:enc establishing an infec-

tion rinte of 100' The mean iross lesilo score was 2.6

an.3 there mean henatocrit value dropped frLon i pre-e-..p,:-.vre

'olume of 13.3 to 23,.7 on ''th day pos t-expo'ure.

Ini convent io nl c*hick.- kn clinii.cl yi:mptoms; were

identical '.ithi tho,- Fseen in 3FF .-hliclkenis (Table I:I.

Infe.: tic.ni r't.c waS 100o1 -nd m-en gross lesion ccore ma 3.1.

T'etnty-tihree out of 104 inoculated chicl:en? died d i.le to

c -C I co..: ii ocis re.is terinri ; a nor t lity rAt,. of 22.11.

Th. :n;c-.l hcn-tocrit v' '. l d'irpped from. a pre-,?xposure

volume of 30.3 to 20.1 on the ','th day.. post-:-.poutic. The

cl inicnil ;ymptconc, it o~-, le ioni in 1r1 t -OCLLii ,-']ii the

p'rec.ice of large numbers of 21ii -eniertion sclizontt ,

c::'e : i"' -i.'- i'dat ion of the cecail mi.c hm:. hemorrhage

on his top ;a-i holon ic :-:imin.ntion confirmed th, preoZ. cc of

.cc-l cocc.idiolis s. dcscribcd 1...' TyL:zcr (1i29) nrd Ty-zzer









2 -C1). 11.T-2). I?' c-c:: im'.'in- a u;-c*. lHL :.n ci.:'Ce (f +r++

,o. +++, cn-..ic.-,.--'i. s .t:-c "c r :c- n *:.:: ildin. iccp ii he -

,i.t.Cl" l-,'ers f tr.h' 91 ill :i b ,."teri3 :c.iil often

'c c n aricr. t r3: a : aci r ..CO il 1 :.co ThE! Coctal J ruiC

ro 'tei t *c ic' t iiitr i ;t,.C i arind t;he 3erii pr -otein rac' t i-:'n, F i n

in ', .t:d a. n.'. nfi. rf,' ,t d .. :' 7enti?,n.r l, ;FF an., b:tct F a: ri' -,

firi- Fn. FFLCO-fr, ? i:.n ar; rEp : rted in Ta tl.e II.

In *:,c.?. ,.:-t ,:'t.nial .i ii 2FF chicleni:: Inre.:1 tei ,i i E. tendl]

Chtre :, ,?. 'n Ltent r-educti.:. n o t cal .i.'ium pnr:. ini

?ouiir; it' :ticil -,l it,,uc r i';.iCti-'n .l a t :-',:-: in 3'ct,:-'ii-,

f .u'n i n-id FPLC-fre.,: .hi.ilcn" -.::p. i to E ten l. u,,,

: pared to io totiLal 2c.rLrI pfrte.-in coiie.it ratic.n of .>-

ini'C cl. r. c'iitr-r:: .













DISCUSSIGII


The cec l. nicroflora vz': found to change in chickens

1 to 35 days of age. Also, changes in certain taxonomic

groups of b-:tteria occurred when rte chickens ,eire e.:por,-d

to standard doozes of sr'orulated occ,'czt of E. tr.-r.lla.

Earlier w.Jorkers reported chan-.:c ini the in-ligenouz micro-

flora dependini'g I'Ipoin the aze, feed, and cn'.,ironmr.nt of tihe

chiil:cns. Cli I~igs in cLrtariin grc'ups of c.bacteria rin the

ceca3 have teen reported in caces of coc.-cidio.is bl,'

Joha.nzson and Snierler (198) and in cases cf "blu.ecomb" by

Taqi ct -.l1. (197l r ) Enterooccci (S. fec-l is. was the

dominant orrianisu of the, ceca of neuly-i-htched clii::'.,

E. coli :'is present in small nurnibers and bec csme dorinriii .

onilv in :uei:-old or oiler cliicken?. The~; results, as uell

as th1e occurrenc-e of larze numbers of L-pctchcCi.lliu Sp. and

.. Poli in chich-:.n aed, 14. to 31,5 d.-iys old, and the con-

stant isolation of r ic' rol.-'s sp. a ree with t'i, re-i.it.-

obt..'incd yv earlier '.o:e:or. in cliic]:, ns (Huhtann :anil

Fet:-.cl: 196.5; Tirimms, 9 3) and in tur!+:' (liani ct -i].,

1970b).

R-a.ll t al. ?17,.,) studied th, distrlLution of tarc-

teori in f,:-c.s of s-. ic and id. R' ifi-- l c,. l, I other

lactose ferriinte..r:;, lactose nonf'-ri'mentern 2, -r.b-r 1 or,-,f.cii








p iCl -:tridium tp., nt er o-:,ci a-l L: toi:-'?i.l : p.

T .-:; co'icl.ud thi3 dijl' b t-Uion ,:' I a.: teri l ,c:-i iS

zinir.- d:n iiin flce a ird c-r1f -n I: miit occcr i a:3 ii.:crzet

mi roc, oi, ':: ri t 2r t i t" a in 1 idi ual cell: i'i .5 iL-..'l- C .

J.loneS an i rift Fih: (1 ?'- r'ep:'rtei that. in ':,i -,i. -ct -: ri

,-' ij o, ie, ar-ni -.a. t .i. iu .' ,) azll., r'epvrtCa

Ize cc,,irr,.ce .t I ll, nic: of LJCtt Ia or th, Wall: i f tNJ

_,:tr'; t rin:t'sti'nal tract id 'cc.1:nts. .olac1 : et 9i. 2.9?,71i

u. i,'.r cc'.- eni.,:']ial ,dilution techn-+]'iqu.: r n:'' d more *.cacia-

tion o r-. ::rnp e an 0a I 1 1nim ? for "I.'-. r' al ,r'OiLpS

.'r :rg 'i Z.ii: T]i. the on c.:pt .[ ba le- ri occu':rri-. in

- ol.'ii in- fe' l :'-n 'les i'.-l i t r ,n- .":troirL :.tiri tract

:.aI'z. n'-?irii' fI.l : ci ic.--l iintrp'el a, i.n c.'f la d- riv, d

*r, c' cnT-'to'nal iilui ti,' fechriu41. ,ifficult ,e _a.s:

Cc i- ,r'matic, r:, '. 0 'C1'. Ui t.u t i .' ;l t1ut de CiC. ii. l.at3 -tnd

not i ri :: re the ,?..a i ual nuc'jat 'f ,' col i e' p, es.er t C. -

t al. I'126 ) r-c p::rt'd 1 .?:.s fui i colat"'in La: ter.r id

*p. from''l SFF :iii after :. igot-ou. : LS t.bif' f rt e n. uc- to

eliIt the nltr'.app:, h .Ct-er'ia. a SirC. tIe diluit ior te-chniqu

h.: uch i:ri'it I l' i i.c th- Lec.lih ique of t :it g

c.lipl.'. usii. .i-. :taai.'d loop I c'..'::'t .pC l a .-t. u1;ei in

this: study. This > :.ir, qu-: h._.: b n u.:.e.7ssf1 i.. *:.mploye-d





api :,c','tll i'-eilt c"'V.'.',ti" ,il chic':".J r i l ir stud '

I'i' e' i .iti i 1 .. .in, ; of Hu-e f'- n,-rcr- -..f F :' :I I ,l 'i .

Tic a r'ar; ccurrCi: of p'i, .: I p rf'im .- hid









thouii,'t ubl. 1 t u.tonu in natrllre, has b- en "ariourly e::plaincd.

L,-v ot -1. C1i 75 ) ,reported tha.,t '. e. chii tC. "r_' in,~_n_')

liis p-reentt in day-old chicli in "inlfected" quarters lut

not in "unintif-ctc" quarters. They established a correln-

tion l.:trc- .', the -:feccts o:f i-antibiotic.s on the o,'ouv- biird

in the prscronce of this or'-.rnism. Hunt.'inirn and F-anLsack

(19%65.1 failed to substantiate thiir report. ;.qi et al.

I19' l b) foundii clo.-ti-rid ia :cCtoce nor nfermeni ter-s an.i.l -

trrai--E :p. present in turI'key. early in life, but: the

or-ganisns decreased in numbers or even irere absent iti older

turls:ey. They believed that such cl'rgaiimc on account of

thnir pticvcnic and in',v:i-;e prcrpertics, are eliminated

by tle drcfn-- nercl'lnisni. of the host, as reprortcd by Dubja.

et 1] 195,) Thi cc-ntenti ion L:eLuV- unlikely sinieiC TinmC

(196l r r-ported r--ular i"olitic.n o:f PE.cteroide. .p. and

,i. welchii ( r"'frill:e .o) from cec.a ,f chici:enz 1c days,

7 ueeks, and 5 months oli. There species there, hc..ever,

absent in the small intestine.

In the prr-enit study, C. nrrfr'in;"-ns uinn i.sol.ted

fron SPF cic::ens frequen:tl' Th e SFF ch irlckenr ii:-r

henlt;,, nn7,r-i no "infec t ion was noticed. They were r-iised

on pasteuriZ'ed fl A aid ,ntr. Al:.- t, "'i il 3 sp. rr'.re

isc.lated from tliese SEP chic,;:r.-,: 1 1 -.: fron conven-

tic nnl chick:ens. Ioroover, itin ,"'reemenr t :.'ith the report

by .nJohl in.-: en-id !u rle i l -d-. ) ini chi"ckcnt c*:.:t n-.-iedr to E.

tn-nallD%, a stilmulttion of ,roJt h of C. pr.f'rt'inr -:: occurred








b tth ,in 3'F and cnre'tit ion l ,l'c;,k, -- :ifter e:pc-'ipo r- L..o E.

t ~1'11i I'l c-onventic.rnal chic.en.: w it cc:- 9l c..:'c: id i : z,

C. fp rf i n' nri I.- re- ul arl,' 1: so. 1 a te in co.nt razt to. rinn-

e:po:ed c t. rol- iri whiL t Ch his o,'D ::nis 11 isZ l ted in

nl:,y 2 c, ,t o.f 3 t ij In 1 of' those t 'l.:il tne :.tIler

cnic,. of the e::periimental group utre J.delibrcite;i. inocu-

lated I itn thi: cr a-nil m. Tnle mechiani ms b. it hit:.h thie

initeztZinl bacteric l populi-p t ioni is caui-, to fi',ctunt-c

t,:cre :not se--,sert ine.d in tnis 1 tudy, but a['rctor-r su'c.i ac

int agon.-,Li m b-eti;:ren b cteria, conpet itin- fol- co:imon

n'utrtients, and *halinr'e i1 pH *-.rd o:.:ia tidcn-reduct io

potential ia :,' pisy an irp:ortpnt :irrt ,Hentg,,z, 19o7, 19or, ;

Lev e- t a 1'5"; iie.'ynell, I0o,,; Schaed.l r -t ., 19o:., .

Thfe pi'v- ent 3stud,' cotrfilrm the ob -.e-Pl';aion :of Jo nhasi'iZon

and Sa-les (19?.?) that :-jrowth of C. r,,tfrirnvrnt in the

cecal flor f citicK: lich. an incre .se durin r infect: ti1

with E. tenr ll a-i ;i.r:.iz,th oi f L ctob'' il, s p. s-hc t .% i

rEd.uctio. In 7-day-, 14-day-, 21-day-, '..-day.- and 35-

iray"-old crilck :en with cecal ?o.'cidi.:sis, a wcuppresDion

c: .:ro.itr- of L--toi : -,.- llu :p an a sti mul-iti:n of .:-roui th

o, r P t-': ides p. C. p rf ri I d .- i : c. noted.

Tlie fatc rl 3 i reccinsibl for i ':urtpr.:.' siont or an n croLic

ZpCr-ies during accal cocc.idio.i- "Jn.:lL .c I iL'L n -'itly

in no rh-,mal, h-ealthy c hici:. ns anrrd 2 t imri1i tion or a spc,:.ies

.*'*hi O _,:-CUrrs only Zpar el- in ni-. Li c-icen:. I1 not:'

;,'t l:t r.in. It. rialy t- that durin.l- thn prl' .r i t:t ani 'o

patent pt.riod .*f cec oal c.:occi io:ic, fa.ctotr :. f itorl'i,_-








-::rotth -.ni nuultiplir at ion or C. -r.frirr-rn ns ma-C tbe re-dlily

avoilibtl'e ;:ith a concc-iiitant reillcl; icn of tilhose fa'.'ol'ii

hjt'Couthl and multip i-i.:t ion of L-,.-n to. c ll sp. A dilff'er-

enc: in -ev.:-rity of tl;h ce Cc-al Ccc.cidio inis syd.lrome was

nrit itd betucen chicl;cni rioncocontaminaten d with C. p-'-rrrinjens

:i'.l chi-',: ns moi oci nrtamn i nateI d n .it ii L ctob. ci ] o llus p. In

Lh-; lforrner, tihe pith llo:i13c 1 r:iani fectations tiere more

sever.: Lhiirn tlh l tter. Al'c., m ie c'er. p'Atiology

du'.elocpedl in chlici.cnr mo'nocontaminsted either with 3.

f'-.'li.-, E. col or B-i-er oid 3 -.p. t ian in those m rino-

c-ntc-r.iir -ted with L- c-tc-o cil. 1lu sp. hen e:.:poser1 to ct -ind 'd

doses of tei-,11 .occCys- s. This inilicate7s that the in.-

Cluence of microbial fior't in the rpotholog-y of cecal

r. occidiocic ci1 epndc .'upon thle p..:ci-e in., o l-.lcd. This ir

illich more cl: -irly ,viid c t ,hen t h: pat lo-l : icnl man ifesta-

tion ini chic !hen: mronoc ont .rli;ri t,.d : iiti h -ir obic feral

.trerptoco.-,c i -ini also tho-.e m.i-noconi tminoted uttI C.

"-1 hi.n c i a it cnpor:d ulth that seen in hiicl:enr mono-

cornt.-ini-terd with otlhi.r specie. of microorcainisms. Little

p th iolo '1y 1 ': ;-cted' in c;hic,!:eiin harbolr-ijn cnly o an er- obic

.:cal s1 tru-pt c. ci nj, i .cul- iticor, o. h:at-kill,'d .. coll,

C. n- .-r 1 nd S. .- c.i di.- not inflr ence the de-

v' el parent of ." 'to ,i cor p-t rli.-r y.

l-thclo.;,: andI clinic i1 ,rptLon: vere neiotliiLible in

chic.:e;.- no:ic'..:.t in.t d i t.h C. I- i.-n "a- d. only mild

to ,n.odcr tc 1 ';ions d ":-lopd i,' chic'l-:-:ns m3nocontanmintedi

ith Pu.- 'pecie Uni -'r natural col lition ic., fungi seei









t': nii:ve little iinflrYuence in the developr ent of cecal co.ci-

dicsis In r le.ie -i ;, o.. .ln-ri -inal n*?!hckcn funr:i occurre-d

on]. intfrequently. Th i mnar' be due to the '- nt a3oni nm

beLt ieen the f iunr arin b ac teri.i a iljici ic 1 iell :nomn in

rilem rbicl ,ic Balish cnd Piill ip ( '1 ) founl thiat ini

the preenc of b'act.ri, C. 3lbi'?ns ij: able to produce

n 'O : l rlo in the p:r. of cliic.'l: en but in the -bs2enact of

Ioti'r ic trf llora, Z. ?1 bic n pi odr .cd C crop I.- 1:i on:. In

noninc:c iulat".) S F chi-cl:~n C. -i, :'i ti us3 more flrequient

tliain in rtioninocul ated ci otvent ional clhi icken. Infet tion

.i hi E. t ri. ll -, .-.lid n':. influence t 0 e ',lroutIh of this fun1 uS

auind an inlibi tic. n cf i touth I L s e.id'ent n in.?e iSoa 111i':c r o

f u'lii in chi -ien: lin.'iinr- ceal cocc.id ioc i3s r:ac infrequent,

bo i h in S'F arid .:onv:e titon l e n"irnonr its.

A ,cripa'i s3.n of the pattiolorg of c '?al c,.cidiozis

in SPF, convent.ionil, oivn pro, 1c .: nt ar i in at d c-i hiceins r'"

in pla:1 ti filml isolator': cl-e.rl s. how:- i l' i-e influence of

tie enm i t--:riezlnt aid micr.flicors in -ecal cc?.:claioie i ,'n-

dr-o e. The- moot o al it rate rom' coccid i' r' i iC aI-2 liFier in

In fie.t-ed SPF c: icl:enii t Isn in i i fec ted. ct en'.:ltion -; i chlick.enc.

T! C-c'u?'rri n e and lthe n ur-.b r ,f :olo.: ie of y..i r.- t.,'

i' is l ; h irnr in .FF cii wienz t!.n in ? ,rn'.iLt ion I .1 clickc-:ri-.

Tfn, ir the onil'.' ubstantial dit ferenrcc in the ,icroif'r.

of tihe-e t',.. gre-oip.. In polyc :*:.'nt :i in *.-d chii l;en.- r-iised

in p .la tic.: i l icolatc ir ( t ri-. r.r t; n i de 1 .-n:i-it d .

"co''v. -r-nti C'nalizCd" ch,.cl::.n:' the- [r' l it,' i- tiLe ini Ilirh

nh-i.-n C r-., r '-i n '- :n ] .. r c -l.i ie.r- rin:-ml:,. of the









T ic roi lorni. A r po A ib.le role for' these orc:'lriiisriT ini cau ing

ae:ith ii chicken: cannot b: ruled out. t-ilichj aid Flililipt

'1966) reported 'va' cul r and r'n-'al invasion by cnterococci

in chicl;env and EDomernutnh and Ci'o:.-. ( 19I69) reported acute

septicemia ofr young and bacterial endocirditis of older

birds, due t.o C.. ,'11s. Dia'rhe:L, lecion- or h:monorrhagi

necrotic enteritis and dc-ath h:avc Lbeen reported in nruborrn

piglets die t, C. prj.rrri nrl n t'rype C infection IField and

iGib.on, 19.55; Barn es and [oon, 19&6 ; B.e.'sel'ind, 196 j).

In co nvention-il chicki-rni the lesions and reduction

in hlenLtocrit n.luez as a renrult of E. tL--n-lla infection

uere more noticeable than in SPF chickens, despite a lover

rate of nortality. Johnson nd Reid (1970) reported that,

in' certain cases, leFrions will be morc severe in ii.'e bird;

thr n ir dead birdc during. an outbrcrak of cecal coccidiosi.-

Trie ire of" hicks to recover 'rori the .-hock due t, the

1 .lr. number of developmental stages within the mucosal

players suffi-ic nl tly c:. l;,' i t.efoie Ecal1 bleeding occurs

may b-: influencinc- the dcath rttt i(E:rtl., 1963 The

i'd. of b -ti.erial species lil:c C. r='frini ecr and q.

fc,--lir in ths- initiation of this tu:pect,: d shock or in

the failure' of chicls to re-.'o'cr 2'!o thi. hock, if any,

1c not k no' i.

.Anothher f,.ature ,f cecal coccidiioni., un-rstricted

h:-,nor agn with ino coio-il :1 ti i".. :el.:urn ,eein in *-'i.ckeznz

harborin, o:inly, :. fe:-.1 i ..,ct r la Im -y hav.' ?, d iniite









P.:.c-l in' '**epin t lpe i 1C -i 'rc .n: f- .t1 rit,:- r''ly-

c.::., t:-.iic, ti: 'n '.lith ,. r..:rf.r_'i 11 arid _.. f,'e'?-.11- ,5r C.



ii ;:.:,-. r::n itch in cl- -i: l : Ecal coc, i.i :1c .- I Lar.-.im

u li 'h Hi: ,.':.rt l' l .'.. i' i i'i L ',." *'.;I I t '.* ' r .,,.',: t-e: :p .: ur-,ll



*.:* thr.- 7th i. p, : t,-c-. :p : : ure ;:. t.-.li -l '-, C i u rr,:i i r

rF Sur.:

TV i i ihiu ci th.-i.l.r io a.. F Irc-'i r b ny i -:'f I i

vari,:,u- t :.,:',cr':,,1 ic -r,-.,.ip: i: r,',i:,;e evi id I.i dhen I 1 *.-?:,-,:.]l



FFV.-Pr, -i- 'r e.- ,':hicr!:,:. i* n :;p.:."_ :,,e, -.i, t L" ,I '.,' ,.'. li':it- : '-

di'i r:'t .1 "c-- i ".,':, tr,. rqilhc..-,.i .m.c-i c l1i i.tr, .:.rf cc .:'e '' i':i .-" i r i

Tie to.:. E. t--.-.,e1 -. l.:. rr .rtali t,',. y.:, r.r d in thcl.: g .rcu .: -.



.il. :i,-. .-le ,:.r r.tul t i- 1 -?.i% C. r i.,c-teri i i zI.:,c; -le-

,l,:,: C llor t ali -.n I oo dy :.c0 1 co nt.en- r i- e n t.-d

,'l:/ in t .:- *?m,:. p? .l:,cOr.nt.iii nat t :1 tl h c .-:\er .a-,i:-j p.,

E. c l : C. r, r'-ir n. nd C. r-a1 .. ,riup m: Cn.-

: n i ir m jat.,. iitrh e. .- t l. ..-].p:.d ild ie .i:.n.-, but

did ct' cm r.oc.-t ii- or bic,.::i-fillej .cec 'Th. .c-.

c aii a' ,c :. c- Al ,,' rt' i tiil .' r1r- r iaii:. 1 ot e 1' .:.:- .

'"-.:: I.. chic :.n t .- .;-ri'," .'.ll- i : _..r. -d ~' i".

siceprt i.-: to E. t 1] tti iiic: cr . ., 2:r t .-

C1. Rc, ,c (1' ,.7'DI, C:. .1-' .t"- Lth- .:.,'-y' it-. iu ,: f F. ,'- i>,11

c, .cy -:' tb'-,rv d : -.pid :,.1 ,;r' ito,.r -.:--.:y r i. ,:,n in ,i-ii .k









at,:ed 4, 5 and ,'ecl.:. rind 's.? suc,:c s Lful e:':c.z-tat ion, in

1 d','- Or 1 ..: .--old c hic!:n 'h-, reaLcr susceptti-

bility of the 4;, ., and 6 iE:-l:-cold tchicl'cns w has thus

?scribed io the -icce..ful r.,-id -.c toit ion of ther

oc:,jcy:t and libcrction of 1aryg nunb-rs of sporociit es

in tlih c-,eal lui'en. Ti' ]o" l- .I.-t:pt i ility of da -,- ld

or w;ee!:-ol.l chickens ;'-- ]il:c,'iS.e ascribed to tniii less

Iucci iful :exY," t'ition t of ocyC'cts as a conz,'equeItn of the

less powerful 'indin~fiE action of thi-: immature gizzard and

,-iearth cf rufficiert tr'p.;in in t'rc new.jl:,'-haLtcl'ed or young

hick:cns. Tryps'in, bile, ind h'.-droa:cn,-ion concentrcration

r' c:orme of the fa.ctors cc.ritributLirin to the e:.:.'s station

(Levine, 1?42; Ikrdda, 1?5,, 10 '., 1960; Hibbert et l.,

1969). Hiib :rt _-t al. (196'?) fo.uid that oc :st s of man:,'

species of E -rin, incliiu lin. t e:--ll, i, f:iiled to -excy: .

if bile alone or t;'r.3in alcr- is present. 'The: also

found that ooc,.:ts of 2. Y.o'i .?nd C. ellir.,Oid .ls re-idily

e:x:c:'st in bo'i,.i: i il .: cc Ot-irni a hecv .' S.ip.iCn ion of

tb-ct'eri : an fun, -i. The cr.-cific action of thie bile :r

ti -e tLa te'ria :-.' f'L.--i in n.? e::c,'Et-tiio n is not :kncl:i n.

S..tnilr 'l -i 1 lt:r "' 'i1..ii of their :: .l l of the s-orn-

lated o:.t:y; ts : p'?ll. e in t'.- ferT.s of .x-3:.osed >cnic?!.ns

"3-s ob etIrv ty LOt -1 L, I1 :-'.. It is pO:; ibll

that enr::,ri' e i!:e Lryp:-in c -n ent ier renl il .: into the

ooc:.-t Ja ,:11 i : r11c ..:' 11,' c!-1 ,: The pre c:nt

cludy in.dic.itcd that n.:-velo .'. iat of coc.:l cocc idio;i3 Ais









r* p rul cf r4pid niji r: _t.r rate of e:-:c::tr: t ion scn tO

:' .r in .l :.'--. id ii '' .:r- 'rcl iT : O--C. ': .i L' t i al

. [.a..: -. i' n T'i .I reI' l ril' i :. i t :cd i'rom cec'- -f

: I', :' i r in. r t;,' l:-pi l li r ., : 1' :c '.::51 C, cI c c idi .-i;

01ii c. C
lic ic -: :'.:'ccc: fril in .:-i"'-cld *Nl'-: r r': T'ncL,:,Ocnt-i !'in ted

i t, t. 1'i In *c',ici:": uI .c: Cold, 1.- z: C'c : f-' .; ul

an -d : ... t t ti1 .-C IT. tc IC MVe C occurred I. d IMi ;; : .

C'e: 1 .- 11 .or both wa h'a rbo' ed in th: c ec: if

the 't '- [ fA' d c .: 1: tat. i 1 : C. rr[: i '. : t.d ith '. I

,d v lic-rie i:t f i- ,di -~: Ii'n th ne:.l- l?. t-:hed 1n-.'n-

t' i lr' : i': t e onl. ti r mi r ,; rga'i- ?i. Fi'ed'ii ri na l ,' '

c. .-li w nd _. *:11.i. It iu .s 1-' foun1 d that the p.-it"ij.-

li:.iLi.;ni r.anif:ct-tin: o f coccait.1i i wtere greater iY C.

:fr i t'!' cif'':' I::c i tc i 7li th either S. re-c:ills orC E.

col i Une her C. r-r 'ri 'e:.7 soids an :hi c:*C'; nation :.f

t'Ie .cUC": .," i t !.ki-n i[ d Ih.sI not be.-:E L t ,iid, ied.

r.! ,-I l t':'c.': r '.:iJLF 1.1 s.hubs qnuent i ber' t r ion of

Sp :.ro-:ie l me ? not be I he -ole r'o-..?.: riich will iAn-



in E. e i 1l.5 in-i' .:tioin H,,rtor' -. iit'i t l1 I I'[ l.

r p:,i't ed c i't'"n l t;: -.'.-- c t ion C '' 'r -i pc' .: trati :ri

of 1 ii. e .t- ?d 3sooc : it. : I ..u. 'C i,:ei: It i 17 :

!',o,-ln t t much o f th-: paI l h e: ,i In *:e.- 1 copii-.3 7i is

due to the m-rr: ...-I .-. o ..Ior "' i L.cr : of' t he la --. -nd

,?,.n tL -. :c. i-iort: r iii-:.,, d- tAr,: lie pi thel iA c 11 ,








cnusing slou-hing of the mucosa and hemorrhage. The role

of bacteria and or fungi in favoring the rapid development

of the enldogenouus Stages, if any, is not kno:.rn.

The pre:~.lt study show-v that typical cecal cocci-

diosis originally described by Tyzt.er l19291 and Tyz;er

Ct al. (19321 failed to develop in chickens raised

bact.eria-, fUmlii- and PPLO-free. ilo clinical symptoms,

mortality' or decrcase in hcuatocrit values fol.louin.- ex-

posure iuth standard doses of sporulated oocysts were

observe d. Lil;ewiise, no visible lesions were noted in

thcse chickens. Chickens raised simultaneoiuly i.nid: u r SPF

and conv:ntionral environments and inoculated urith the same

.tandnrd dose of sporulated ooc.sts o:h hibited the classi-

cnl ecal coccidiosi- syn.rome.

Thlrie was also a marlk:ed difference tbtwuen bacteria-

free and SPF or convent ional chickens based on hlistopatho-

logic findings. Ceca of the SFF and conventional chi]:K.

inoculated with E. te'c!ll' oocysts and necropsied on the

.th day post-e,:posure showed e::tensive denudation of the

nucosa, h.morrhai-rs arid large numbers of 2nd cn-neration

shiont.s. In severe cases, the hesmorrhLe:ic and tisCue

dfmnac even reached the muscularz laye-'ri, and the entire

mucozal layer ri-' seen to be? stri ppe do'.-n to the ba.c- of

the submucosa. Large 2nd generation scni-ontZ lying mostly

free and a feu within their host cells -ere the prc-ndmicant,

cniogenous stage seen in such c: es. But in the ceca of








b c, te'ri:-, f' i ..i id PPLO-f 'r e -!i,': c..' .: e i:.ci.-.o t c E

t-.: 1 o.o:.'t .nt m l necr. ': .i.i on l 3th ;, o :t-c. t .cr. ; i -e,

o e::L:n 1 :.': di iNi-:t n n. ': i '.a, hi .:moirl'i c. '. i ri e

." !i.' i t -' _irt _n 'Ji:c rr j : onr seeri. 'ThE : ro; osai lay2r

iow.': r,-]. r c: le." i t ..;t and c.t fic.: r '. i -zl '. L ver'C Llni'ld iaSl a.-ed. ;

ni. -iIl -e :nd ci.-t:i---cn r: chi onit uere l o-:n. A -?ti en-

- 'lo:.-:rn, ti: t.: ,.L : ideni ified ?' iTim a tui LZe chi-. .nt : it re

::ti.:ti in ti l:. eitnelial c-llz ; f the r ucoz' : .zpeciall',

1io.. lliii-Inv t e giitd f[undi Tiii location cf th a.e;.,

.i'u--e:t- tin t ti--,' a n be t: l t 'c -m r ti i on :' hi:ont -

dev.eli. nr-, .cz; l7 ,l/y. liti- ,g t that in t.:icteri;.-,

'ifn i- -id P LOF' -fL*C Iic!':n the ic,:rree .rid rate of

lt .' r]n t if ,'.d.;e o. cta';-e. a .e m rarke-1.dl., inhi biLed.

T i' i nhil bit i':.n i d '.': 1 1:c ...i If cir-cd c T ta.;e ,

e cz,-..; 1 -iy t he1 p. t h- :7 .ni si ai. 1i cno ,'- t-]nic 2nld ne r -ati. ra n

' iizo.n m.;.' afM.i.3 pr 'otEctiion 1. tc host aind the pa- ti,-

1.'"-icil anifer t-t i:onc: ar.: no:.t .I-.-elc.pel. In r-act, the

ta:.ter i -, futltti- and FFLO-fre *:nhiic:enc e.;,i to:- L'e-have

-i. Sn b ariLicu 'line t Lhx a c : cat i I ? i '- 'c-l in the

c t:t inc; eicic:,e;' r;t .e: e ro n i the Pr:' c. lls

: t -'..-:'.:'- t i o Wi. I-i: t c h:1ii A i-t:e dii 'i elorp-

[ 'ent .Cin Lo i a t.- r ri i rd d. el :i ,1 F1. t 1C h ,

11 ti,; .li I0.. ;" .8Kl-0 bTo,.c: r,_-- c_ ;lilcl'...ri., O".at ': 'cd a

12-1 5 h utiL ..cl ay iii t': .".Fpe arnc'.- of 2nd ere r -t t i.:ii nero-

7:-, t i r tth C'e,- of 'Lt_ Cri .'- -f i C i_ C' : ' I H-:Iie er,

tie,' f i.i.d "o il'f.ren iti 1.i1: ,cOrP-e Cf i *n.'ction: in








ba.tc -ri.-free and c ioventinal chickens. Thic nact

experinenital pi'occdur'cs and re.-ullt are not conpl.ctly

I:noim to compC, arec their' rc.sults i n ai th,: fr tIe prPe ent

t udy.

!ait:enbach et -l. '19'66) lescrib'ing a method for

.terilizirin cocci.dian c.o,:;'sts employing Clorox: and uil-

i uric ,ci -dic}ror.ate solution, indicated that theY' coulll

prc.duce c,-.cl c.ccidiosis in "g iotobiotic chicl.ens."

Since the e:act nature cof their "gntol ,biotic c;:ici:eni"

ha:r not been indicated nor has the description of th

experimental pr.ce.iure amid results oft thc ji i o.rk co.en

given, it iZ nrot pozcible to compare tlecir r-esultz ritl

the present work. Thcir ,orl; u-. di:i.il Lt o obtain

sterile o,.cyts for cultivation of the pI.ra itet il tircue

cult ure.

ilybcrg an-i :narpp (1 9",3, 1970b), e.:mining the

Docsts of E. tein.cll by reans of a scannruing, electron

microscope, reported certain structural alterationci- of

oocy,'s:ts. ioiLne an:l Honii l. 1'54' ) report .t that rhen

c:oc;,'st are tre-iteti wit.n sulfuric acid prini, to soliupr

'hy,'p', ilc'lori te, the outer layer lo the o-cy:Lst '-311 L .r..:-r

u'i'.i:ll'.d and apparcntl,' elev:,tet ai.- fi-cm 'he' inner

l.-,ver. Emploc.yni .,e t of C -uch .tricti.- 1] *.' 1 t.i-r'e oo.:z.' t

miiht influence pailtho.enesis 2i11 d.:'eve:lOFpncct c.f r.i'-aw-.

processe: of r.c c cidio i In tr...., t-h u.e of C.

per.-cetic acid for surf'.:ce stcriliczti.on of in.-.:rt itlA'teo








*:-.-e- "id ooce yct; his b en found t c. be- .'ry -,' rfect i. 'Doll

-. ,'" ,:; i ', .;1.,' -t aI. i':,,.. ; He- dc, e t l. 1? '1 ;

SprinT -r et :1., 1'1,0). lUlnd r t le lii hlt iicoi' zc,.op'e, no

diTi ', ,r chTi e t :, the oooy,'st 6.all I -I -:-. n I 'hen p':ra-

: -t '" aci d Ui2S LIed ri' ul.ri'I i e tCr' iii t i "ton an, t hlere f r'e

it is a': urmei that p r:icete:ic acid i i'ot it tinjuri'i t. the

co..:zt iIll e- n i.;. d accordinT- to the mTctlod of Doll

-'t i I i'?1 .1 and thi? met:lid i ui.: .i. duirin.-" tlo !..r-esent

: tLid:,. Th'- l'3t- bt irined in the pI e czci .tii .-l: can be

,ompIP-r'E-d t-: tho- *btairnci by Vic: anid burns (19 .6,

quoted b,- il-d ._t ,I., 19-.9) 6..ho b) zerv.e no m rc.t :li t;

in, bactcri C-fr'lee chi-ck: rthe-i e.po?:ez: tc E. te n.i11- c,.cys t

anid by Kem t l. !19'71 .'i h reportedly d l :'ini of the

.- '.-- .p r t of tlhe iendo;:'-n .iOu t '-e in rO ii-fr'e chi. 1 : .

In t pr trt otudy, th ere '.'a: also 'i delay in tie de-

'*:lcpmri:-t Of enl.c~ enou sta e.. b rv a r and tli: d.- lay I.i:'

be b ner .-f i ia I allc.'inri tai nocIt- to de.~'c ln 1 i't'ficilnt

r e.7- 1 sTr-ance and t h-reby inhibi t oti iie.'elo'mcin, of clinical

symptom: -ni O:th er itit'e tati:nt i : of th: dizense. It, is

=.1 o pcs.ibl that thie d.;.velopI nt [" the- Iir-: 2rnd

*.tenerati:rn s.cI..nt i" di l.ia:y : ,,,'r inibit. ed by the

ab.nce ,:'- mi L rcr r ill i- d to inin Al *.: .elor:nit

ofr, piti.holo; :, Inh .:.i t ion I -' de :c.'ll 1 -.: n t :i'id developme nt

,oi i.e r n e ii br oi-i' rln!-atc p- r" it : e bCein fo;.iti, in

Ci'n-iri'e h ,ct includin.- t ,i' C i: 'n (Il, 1i 1 '59

J.Tohnzon, Sl"'] l. Spt ii,: r' :.t 1. I1 i: rep r.ted .ur. ivinl









of H. rlm 12l'-: idi s r ithou1 t d1 Lca- e product ion in l.-rot abio-

tic hosts. Confirning the -synerzisftiic role of certain

bact ri'l specie- especiallyy C. crf r ein 1nr) and H.

melea7nri1- in the p'a.A-ct ion of infectious eiitroCiCpati-

tis in turkeys reported oy Br-dley' -t il. ('.19l4), Springer

et al. t:o0)) established thit tle role cnf bacteria is

more vital for the -urvival of H. rcl1 i.?nr'm-r l5arv\e Lhan

H. mpulea?'idiz in the pathogen-eis orf infectious entcro-

hepatitis.

In E. tenell? infections, the e:::a'ct role of the

bacterial sppccies i, not no-,n, but the proec-n:. :tudy

indicates that for the dizesce ;yndror.ie to do. clop in

full, certain bacterial p e-cies ar-' e:7ccential cince no

mortality or gross lesions riere teen in hosts frec. of

any detect ble ,org'-nisjms Johncon et -l. (1l67) and

Roh)ovsl:y, andl Gricpe.ere (1t'67) found fc-line infectious

enteritis in the germ-free cat to be a mild, nonfatal

disease .:onsist.irg of leukopeni?:, thymic atrophy ani

lymphoid depletion but I ithour morphologic inte'stinal

lezionz. On the contrr-ry, CFF -cats developed clinical

si:; : f c typical inte'.tinal invc'lvenmnt a.: rcn in frani:

infe'ctio.us entcritis. Sincea- L- ,e cecn of th. ':.hick:ns con-

tain the maior porl.t io of the lroii:.' ntin.l flcra

ITimrs, 19r,8), E. tpn.'ll- may have d.c,.lo;-pi n apparent

obligate rclatiorisiiin .'ith c r't-ain m-: i' rs of this micro-

flora for pat hoseresis to commencn.e a,.-. this z: lntion'lhip

would have influenced the over':ll host-parasite relationship.








T)r c? ,t i l i. n to f U:i i .r .u 1:. : c iO-7 r th

'- 1 ? :. -,ci'iii r',r i l n prod 1ucin c* 31? c. i1 sic s r.;'': i 'i i ir O I.r '

r I' r- i po:cl.-' d tv ,' t i 1 .3 '). ii- n f'". I p 'rt,:-id

IL., 1 TMrerCAc; f c'31 eoc;idiosis to be Aimi3r' toi





;io c' c : th ii .i core '' anid h: rrh-A e. ich-au:z.in .and

-am le"PH u1 .I ;Di: r'port1d a po.:i.ibe irrvolv'.M.nt Ofr

E ,ccli N the tii.._y c, 1e?? l ,co- i.4 iSi Th,?ir

it .dy 1 r -i,. th e p ,- .,-: tt ..Id :hi i',.d1 tIh?- .c. if r_'.. 3.:-

prre>-ut i 11->g- fi.WiriC.7P tIhrir.O Iirut tIm c* ur'- f cc-c0al

-".:*:idlio i- Th' ;y, m imed that thel r.:duced z v:-rit:.

in oile..1 :h c c di.P: to' 1 c: li f rm c-cnt ih thL-'

eum. The' rc uc id -'verit of ,e ai ca cidiA':'s. sr

'n da4 '-o,.i ohi,":en- ?, .lsc be Ikt tc t.hc- Sa-,T- reason,. '

















Tvpical c -1..1 cccidi-,d L did not div-lop in

b.?ct-erii-, fun.gi- 'ni FPPLC'-rfr.c: -iiclkenc iinoc I'i ated uith

r .t:niidard doie of infective ooe.'zts : .f E. ten- la..

Hodi- v.r, rsnail nuntbert i- t.'.1 ll] I were able t c dev lop

at 2 slower rale in tir.-teri a-, funLgi- .anr I PFLCO-frIe-

chickeins as co mpriF:- '?e 'ii th~ r1 te i se r I in co inv'.nt i 'onal

c'ii .c!:c. In chicl.n'' n-once.jt- i .ated ulith C. perfrrin-

g;nc, . fc-alis, E. C,.li, B t- r,-,ii-sa sp., L[.T. toh- ,',11.r

:p., i,'nr ,p. or_' C. :--.1-i.-' n ..:pal r. ti,: E. 1 i- 1 1

oocy;,ts -res'ulted i nnii c .e l cocc-:idi 1i C.:c.:l c, ci-

di'.si i; Ch l'iii;crin c.l '- ntamin-ita d with bacteria ia.

conrtpani" blc :.ith th:it .- n .en in conv.-Ltiional or SPF chickens.

Hi lCher 1mortalityv bun lot.,-3r mein ir,..ross l,:ii.:'ns dLie toi

coccidiosi. .j..re seen in SPF chic!l.ens e::poscd to E. teni-11i,

compr -'i-,.--,d to t'.i in i ni i-'trio il chi.-l:ens.

Iridi-enou'i; i'l 'ra n cf tlihe Cc'~. i of, chic., ei- oLI various

ag im E nor i.li nt i.:.l. Tn v'.'r V'.i..n: chi*,':en.; t(lu to 1

ti.el; of o.. ., rnt. ....;.'c i pr. '-. in tile coca while

in ol.-- r cl1hi.!::.n3 4 t- : -- L -- r oD, -.-.-. 1 s Sfp.

Dro',e o- inr ted .--t -, '; -- c. L-.-'t .. i llI' p.

and S. r I -- i : '-.r' p r.- l -C 1 .ir 'i fr.'ri co.:'.'e tiornle

chlic.-n:; a,:t 1, 2, ', 4 i "':c ks f a; I;oliation of


3Ui:;h',RY AIHD COCLU-IJ.CIO:S








C. p'-rrrijL' ''-". L in'i.' ''iuentl in Ci',V.?in t i"n'al chi'?l'-ien ,

but freq.,ent in SFF chickEri. IIc- ul rticon o[" chiickens

with E. t --n-l 1a rezilted in ?t imulation c" i'.outh ,iof

C. per. ri' -?n-Z both in conventiouial aL-'Ld SPF birds. Ail C,

l.r-.:g nioLmmbci-- of E. coli and .ct='roridee.z p. but low

nurimber.: c' L" nctbcLa1C i l 4 p. i.cere ea-n in the ceca of

inrect'.:-d chicil:eLn-. Tie d.it, indict ate that thel in.dii ennc.Z

bacteria s-id in r'apidi dicve lopnernt cf endoencu: : t age'r c:,

E. tenelli and prolducti on of *', p i C1 rec-dl rcciiiosi .





























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Full Text

PAGE 1

BIMERIA. TENELLA : COMPARATIVE PATHOLOGY AND LESIONS OP EXPERIMENTAL INPECTIONS IN BACTERIA-PREE, SPEGIPIG PATKOGENPREE AND CONVENTIONAL CHICKENS By Ghittur Venkitasubhan Radhakrishnan A Dissertation Presented to the Graduate Council of the University of Florida in Partial Pulfillment of the Requirements for the Degree of Doctor of Philosophy University of Florida i971

PAGE 2

To my parents Subhalakshray and Venkitasubhan who inspired me to pursue a scientific career.

PAGE 3

ACKNOWLEDGMENTS The author expresses his appreciation and deep sense of gratitude to Dr. Richard E. Bradley, Sr., chairman of his Supervisory Conmittee, for helpful suggestions, counsel, and encouragement throughout this study and during preparation of this dissertation. He is also indebted to Drs . G. T. Edds, E. M. Hoffmann, G. C. Smart, Jr., S. G. Zam, and Mr. P. E. Loggins, members of his Supervisory Committee, for advice and guidance. Special thanks for help in many ways go to Mrs. Beth Presnell and fellow graduate stu Mr. David J. VJeiner. Mr. Louis N. Ergle and Mr. James E. Dickinson, Jr., provided technical help for ;;hich the author is indebted to them. Grateful thanks are to Miss Jean Barry for special technical help. The faculty and graduate students in the Department of Veterinary Science have also been helpful in many vmys. The author wishes to gratefully acknowledge the financial support of the Department of Veterinary Science in providing a graduate research assistantship vjhich supported him throughout the study. The research was carried out as a part of a major project, Hatch 14-19 (W-102), entitled "Biological Methods of Control for Internal

PAGE 4

Parasites of Livestock." He is greatly indebted to his v;ife, Jaya, for her help, encouragement, and understanding throughout the entire course of this work.Thanks also are extended to Mrs. Joyce Jarvis for typing the manuscript IV

PAGE 5

TABLE OF CONTENTS ACKNOWLEDGMENTS iii LIST OP TABLES vii LIST OF FIGURES i^ ABSTRACT ^ INTRODUCTION 1 LITERATURE REVIEW « Eimeria tenella : Life Cycle and Morphology 9 Pathogenesis of E. tenella Infection 15 Pathology of Cecal Coccidiosis 21 Microflora and Hosts •• . • 26 The Germ-Free Chick 2u Pat'-n -enicity of Intestinal Parasites and Par. Ibism in Gnotobiotic Hosts 3^ NORMAL MICROBIAL FLORA OF CHICKENS ^0 MATERIALS AND METHODS ^^ I. Production of Gnotobiotic Chickens 4^ II , Production of SPF Chickens '1-5 III. Production of Coaventional Chickens ^6 IV. Source of Eimeria tenella ^7 V. Pathological Examinations ^7 VI. Bacteriological Procedures ^9 A. Determination of microbial flora of the cecum in conventional chickens 49

PAGE 6

B. Determination of microbial flora of the cecum in germ-free, SPF, and conventional chickens , . . . . 51 ?II . Packed Cell Volume 51 VIII . Total Serum Protein 51 IX. Serum Electrophoresis 53 X. Exposure to Bacteria and Fungi 5^ RESULTS 55 Development of Microbial Flora in the Cecum of Conventional Chickens Inoculated with E. tenella and Uninoculated Controls 55 Bacterial Isolates in 3-Week-Old SPF and Conventional Chickens Inoculated with E. tenella and Uninoculated Controls 56 DISCUSSION .' 6: SUJIMARY AMD CONCLUSIONS 79 APPENDIX A 82 APPENDIX B ; 120 BIBLIOGRAPHY IS^P BIOGRAPHICAL SKETCH 1^^ VJ.

PAGE 7

LIST OP TABLES Table Page I Primary Isolation Media Used for the Recovery and Identification of Bacteria and Fungi from Ceca of SPF and Conventional Chickens 52 II Cecal Flora of Young Conventional Chickens Exposed to Eimeria tenella 83 III Predominant Bacterial and Fungal Species from Chickens Shovfing Lesions of Cecal Coccidiosis and from Nonexposed Conventional Chickens 8^ IV Pathology Due to Eimeria tenella in Bacteria-, Fungiand PleuropneumoniaLike Organisms-Free Chickens Raised in Plastic Film Isolators • 86 V Pathology Due to Eimeria tenella and a Single Species of Fungus in BacteriaFree Chickens Raised in Plastic Film Isolators 89 VI Pathology Due to Eimeria tenella and a Single Species of Bacteria in Chickens Raised in Plastic Film Isolators 90 VII Pathology Due to Eimeria tenella in Chickens Raised in Plastic Film Isolators and Polycontaminated with Bacteria and/or Fungi 9^ VIII Pathology Due to Eimeria tenella in Specific Pathogen-Free Chickens 99 IX Pathology Due to Eimeria tenella in Conventional Chickens 105 X Occurrence of Clostridium perfrinp:ens in Three V/eeks Old Specific PathogenFree and Conventional Chickens Exposed to Eimerj.a tenella 110 Vll

PAGE 8

Table Page XI Mean Packed Cell Volume (PCV) of Bacteria-, Fungi-, and PPLO-Free, Specific Pathogen-Free (SPP) and Conventional Chickens Exposed to Eimeria toiiolla . . . . 112 XII Total Serum -Proteins and Serum Protein Fractions in Infected and Noninfected Conventional, Specific Pathogen-Free (SPP) and Bacteria-, Fungiand PPLOFree Chickens 118 Vlll

PAGE 9

LIST OP FIGURES Figure Page 1 Geca of 3-week-old specific pathogen free chicken exposed to Eimeria tenella , 7th day post-inoculation. Macroscopic grading of the lesion ++++ 121 2 Photomicrograph of a transverse section of cecum of 3-v.'eek-old specific pathogen-free chicken exposed to Eimeria tenell a shouing denudation of mucosa, and large 2nd generation schizonts. Hematoxyli.n-eosin stain. XI25. Microscopic grading of parasitism ++++ 121 3 Geca of 3-v.'eek-old bacteria-, fungiond FPLO-free chicken exposed to Eimeria tenella . 7th day postinoculation. Macroscopic grading of the lesion 122 ^ Photomicrograph of a transverse section of cecum of 3-^'feek-old bacteria-, fungiand PPLO-free chicken exposed to Eimeria tenella shovjing a single coccidium and no denudation of mucosa or other tissue damage. Hematoxylineosin stain. X400 122 3 Geca of 3-vreek-old bacteria-, fungiand PPLO-free chicken exposed to Eimeria tenella and Clostridium perf ringens . Macroscopic grading of lesion ++++. \ 123 IX

PAGE 10

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EIMERIA TEMELLA : COMPARATIVE PATHOLOGY AND LESIONS OF EXPERIMENTAL INFECTIONS IN BACTERIA-FHEE, SPECIFIC PATHOGENFREE AND CONVENTIONAL CHICKENS By Ghittur Venkitasubhan Radhakrishnan December, 1971 Chairman: Richard E. Bradley, Sr. Major Department; Animal Science In order to determine the role, if any, of the indigenous cecal microflora of chickens in influencing the development of and disease due to Eimeria tenella, the pathology and lesions follov;ing experimental inoculation with a standard dose of E. tenella infective oocys'ts ii bacteria-, fungi-, pleuropneumonia-like organisms-free (PPLO-free), specific pathogen-free (SPP) and conventional chickens vjere studi'ed. The dominant microflora of apparently healthy conventional chickens and changes in the indigenous flora following infection with E. tenella xv'ere studied in chickens aged 1, 7, 1^, 21, 28, and 35 days, using standard microbiological procedures. Experimental exposure was carried out by oral inoculation with 100,000 surface-sterilized E. tenella oocysts alone or combined with single or multiple species of bacteria and/or fungi. No clinical symptoms, mortality or gross lesions

PAGE 11

were observed in a total of 32 bacteria-, fungiand PPLOfree chickens inoculated vjith E. tenella alone. In these hosts a retardation of the development of the endogenous stages of E. tenella vias evident. The presence of a pure strain of Bacteroides sp. , Clostridium perf ringens . Escherichia coli . Lactobacillus sp. or Streptococcus fecal is or single species of the fungi Candida albicans or Mucor sp. resulted in mild cecal coccidiosis following inoculation vjith E. tenella . The typical cecal coccidiosis syndrome developed in chickens harboring 2 or more species of microflora, viz. , C. perfringens and S. fecalis ; E. coli and S. fecalis ; Bacteroides sp. , C. perfrin,°:ens . E. coli and S. fecalis . In SPF chickens, typical cecal coccidiosis developed following experimental infection, with a mortality rate of 3Q% and a mean gross lesion score of 2.6. In conventional chicKens, the mortality rate wap 22.1;'^ and the mean gross lesion score was 3-1. C. ^ ^fringen.v j.s isolated more frequently from noninfected SPP chickens than from noninfected conventional chickens. A stimulation of grovrth of C. perfringens and coliforms occurred with a concomitant reduction in the grovrth of Lactob.?oillus sp. in SPP and conventional chickens suffering from typical cecal coccidiosis. The results indicate that certain species of the indigenous microflora of the ceca are essential to produce the typical cecal coccidiosis syndrome, follov/ing ingestion of E. tenella infective oocysts. XI

PAGE 12

INTRODUCTION Eimeria tenella [Sailliet and Lucet, I89I; Protozoa: Eiraeriidae] is the most common and pathogenic of the 9 species of Eimeria described from the chicken (Callus domesticus ) . All the 9 species of Eimeria occurring in the chicken are intracellular parasites of the epithelial cells of the intestinal tract producing the disease knovm as coccidiosis. Coccidiosis is a disease of great economic importance to the poultry industry throughout the world. In the Un' '"'' States alone, a total loss to the poultry industry of $34-, 85^, 000 was estiraatvjd during the period 1951 to i960 by the United States Department of Agriculture (1965). Of this sum, .^^15, 123, 000 were attributed to mortality and ^19,731,000 to morbidity. In 1966, the expenditure for coccidiostatic drugs in the United States v;as estimated to be between $i^0, 000,000-.';i;50, 000, 000 for broiler and laying flock repla-ceraent chickens. Acute coccidiosis with a high rate of mortality is regularly associated with infection due to E. tenella and since the lesions are confined to the ceca, the disease is often referred to as acute cecal coccidiosis. Young chickens 3-8 weeks of age are highly susceptible to this disease with a peak susceptibility at

PAGE 13

about ^ weeks of age (Gardiner, 1955)Several other authors also suggest that young chickens are more susceptible than older ones (Tyzzer, 1929; Karmann and Presch, 1933)Conversely, many others suggest that the reverse is true (Tyzzer ejb al. , 1932; Mayhew, 193^; Jones, 1932; Horton-Smith, 19^7). It is important to distinguish between susceptibility of chickens to clinical disease and susceptibility to ccccidial infection as measured by oocyst production. Based on oocyst production, older birds are more susceptible to the parasite than the younger ones (Rose, 1967b). This is due to the higher rate of excystation of the oocysts (Doranand Farr, 1965; Rose, 1967b). Older birds are also susceptible to clinical infection but the rate of mortality is usually lov; due to an apparent acquired immunity after previous nonfatal exposures. Levine (I963) suggested that the acquired immunity is often not absolute, but generally only a condition of relative immunity. One-day-old or l-v;eek-old chickens are less susceptible to the infection v;hen compared to chickens 3-5 weeks of age (Rose, 1967b). Thus the severity of the infection and disease in chickens under field conditions is related to age and breed of the chickens, previous exposure, and the degree of exposure to the infective stage of the parasite. At the present time, coccidiosis is controlled by the routine use of various coccidiostatic drugs. For practical and economic reasons these drugs have

PAGE 14

to be incorporated in the feed or drinking vrater of the chicken from the day of hatching and continued throughout the life of the bird. This continued use of drugs has resulted in interference vrith immunity (Davies and Kendall, 1955; Reid, i960), side effects such as reduced fertility (Joyner, 196^), and development of drug-resistant strains (McLoughlin and Gardiner, 196la, 196lb, 1962; Pellerdy, I96I, 1962a, 1962b; Gardiner and McLoughlin, 1963; Vegh, 1963; Joyner, 1970; McLoughlin, 1970). Moreover, drugs presently available do not offer effective protection against all the species of Eimeria parasitic in chickens and most of the current broad spectrum coccidiostats are not suitable for prolonged periods of use in chickens intended for human consumption. In spite of the high efficacy of modern coccidiostats, outbreaks of the disease may occur (Joyner, 1970) due to high levels of contamination in the environment, "reduced uptake of the drug or development of drug-resistance, and a high degree of susceptibility (Joyner, 196'+, 1970). Under natural conditions, cecal coccidiosis occurs through ingestion of large numbers of the infective stage of E. tenella called sporulated oocyst. The unsporulated oocysts which are formed in the epithelial cells of the ceca are expelled with the feces. They develop and sporulate on the ground if conditions of oxygen tension, moisture, temperature, and other environmental factors are suitable.

PAGE 15

The sporulated oocyst, containing 8 sporosoites, is the infective stage. The first step in the pathogenesis of the disease is excystation of the ingested oocysts. Rose (1967b) found rapid excystation of the majority of the oocysts in highly susceptible chickens ^, 5, or 6 v/eeks old, v/hile less successful excystation and lov/ oocyst production occurred in 0-3-*^esk-old chickens vrhich are also less susceptible to cecal coccidiosis. She ascribed the reasons for very lovf percentage of excystation to immaturity of the hosts (vreak action of the gizzard wall and sub-optimal concentration of tryptic juices). There are a combination of factors necessary for excystation such as bile and pancreatic juice (Levine, 19^2; Ikeda, 1956; Hibbert et al . , 1969). Our knowledge regarding the role of intestinal flora in the initiation, development, or severity of cecal coccidiosis is limited. However", it has been reported by Johansson and Sarles (19^8) that during E. tenella infection the grovrth of Clostridium perfrinficens is stimulated while grov/th of Lactobacillus sp. is suppressed. The cecal bacterial flora constitutes 90/o of the total gastrointestinal flora and is of great biological importance to the health of chickens for synthesis of certain vitamins (Coates et al. , 1968; Timms, I968). The effect of normal bacterial flora on the biology and immunology of the host and possible relationship between

PAGE 16

this flora and certain diseases have been investigated by many workers. Phillips et^ al. (1955), using germ-free guinea pigs, proved that presence of bacterial flora is essential for survival of Entamoeba histolytica and pathogenesis of amoebiasis. Based on these findings, V/ittner and Rosenbaura (1970) studied the role of bacteria in modifying the virulence of E. histolytica . Bradley and Reid (1966) demonstrated a dual etiology involving a protozoan ( Histomono-s meleap;ridis ) and a single species of bacteria ( Escherichia coli . C. perf ringens . or Bacillus subtlis ) for infectious enterohepatitis in turkeys. Several other studies have been made on the role of bacterial flora affecting the course of infection in infectious enterohepatitis (Doll and Franker, 1963; Franker and Doll, 196-<, Reid et al.. , 1969; Springer et. al. , 1970). Hegde et al. (1969) studied the pathogenicity of E. brunetti in bacteriafree chickens and shov/ed that the parasite can develop and produce disease in bacteria-free chickens. No substantial studies, however, have been made as to the possible role of bacteria or other microflora in relation to pathology due to E. tenella using gnotobiotic (bacteria-, fungiand pleuropneuraonia-like organisms (PPLO)-free) chickens. In preliminary studies, Clark et_ al. (I962) found no difference in the course of E. tenella infection in bacteria-free and conventional chickens. There v/as, however, a delay of 12 to 15 hours in the appearance of the 2nd generation

PAGE 17

merozolte£3' in the feces of giiotobiotic chicks. Visco and Burns (I966, quoted by Hegde ^ al . , I969) reported a close relationship "betvreen the host microflora and E. tenella in the production of the cecal coccidiosis syndrome. As Hegde et al . (1969) reported, "the effects of the bacterial flora on the pathogenicity of this species remains unsolved." It is not clear v/hether the bacterial flora hinders or aids E. tenella to initiate and develop the disease entity. The present study was undertaken to find out the influence, if any, of the microbes normally present in the ceca of chickens in the development of cecal coccidiosis, by determining the ability of E. tenella to produce the specific pathology and lesions of cecal coccidiosis in chickens harboring no detectable microorganisms and by comparing the pathology of experimental infection with E. tenella in bacteria-, fungi-, and PPLO-free chickens, specific pathogen-free chickens and conventional chickens. To prove Koch's postulates v;ith regard to cecal coccidiosis, bacterial isolates from conventional disease-free chickens and chickens showing typical lesions of cecal coccidiosis were also compared. Bacteria-, fungi-, and PPLO-free and SPP chickens were inoculated with standard doses of E. tenella sporulated oocysts isolated from naturally-infected cases, either alone or combined with bacterial species. Any organism or combination of organisms capable of producing cecal coccidiosis in gnotobiotic chickens were then

PAGE 18

isolated and the isolates used for further inoculation of conventional, SPP and bacteria-free chickens. The Icnowledge of the interrelationship between the normal microbial flora of the ceca and E. tenella gained by the present and subsequent studies may ultimately lead to better means of control of cecal coccidiosis.

PAGE 19

LITERATURE REVIEW Elrnerl a tenella [Railliet and Lucet, I891] is a protozoan belonging to the Family Eimeriidae, Glass Sporozoa. The term "coccidia" is generally used to describe species belonging to the Family Eimeriidae (Becker, 193^). During the last 10 years, studies on the fine structure of coccidia and related groups have revealed a great number of nevj similar structures namely the pellicle, the polar rings, the subpellicular microtubules, the rhopteries, the micronemes, the micropore and the conoiu.. These fine structural similarities are considered as indication of a close relationship and Levine (I969) proposed a slight modification of this classification and Scholtyseck and Mehlhorn (1970) have discussed the problems of taxonomy of Sporozoa. In the domestic chicken ( Gallus domesticus ) . 9 species of the genus Eimeria . E. acervulina . E. brunetti . E. hagani . E. maxima . E. mitis . E. mivati . E. necatrix . E. praecox . and E, tenella, have been described as parasites of the epithelial cells of the various regions of the intestinal tract (Blester and Schv/arte, I965). E. mivati is the only species which may be found in several regions of the intestinal tract (Edgar and Seibold, 196'i). The 9 species can be differentiated by morphological characteristics, sporulation time of the oocysts, developmental

PAGE 20

features, localizationin the host, and degree of pathogenicity. Of the morphological cho.ractors, the structure of the oocyst is usually used to identify the species at least within a given host (Levine, I96I), but oocyst characters alone have only limited value in differentiating species of Eimeria (Horton-Smith and Long, 1963). Among the many means of biological differentiation for Eimeria species, the location of endogenous stages in the specific region of the intestinal tract of the host and species specific immunity are of major importance. Each species shows a marked regional specificity (Tyzzer, 1929; Tyzzer et. al . , 1932 ; ' Herrick, 1936) for the development of endogenous stages. Also infection v/ith a species results in immunity against that species but not against others even within the same host. Hence cross-immunity tests can be used to differentiate the various species of coccidia (Tyzzer, 1929; Tyzzer et al. , 1932) but the specificity of acquired resistance may not be rigid. Rose (1967a) found cross-immunity between E. tenella and E. necatrix v;hen using sporozoites of E. necatrix to induce infection in the cecum. Therefore a combination of factors is always used for species identification. Eimeria tenella : Life Cycle and Morphology Acute cecal coccidiosis in young chic;:ens is regularly associated vfith E. tenella and this species is the most common and most pathogenic of all coccidia found in

PAGE 21

10 chickens (Davies et al. , I963). Tyzzer (I929) published a detailed description of the morphology and life cycle of E. tenella v/hich has been confirmed in all details by subsequent investigation (Edgar, 19^1). Like other species of Eimeria, asexual' and sexual generation occur in the same host following ingestion of viable sporulated oocysts through food and/or vmter. Through a process of excystation, sporozoites escape from the sporocysts and oocysts, but the factors contributing to excystation have not been definitely established. Studies by Levine (19^12) and by Ikeda (1955a, 1955b, 1956, i960) revealed that pancreatic juice, in particular trypsin, is one of the factors responsible for excystation. Goodrich (194^) observed o.i^ escape of sporozoites through any available fracture in the cyst v/all 5-10 minutes after the cyst wall has been placed in a 5f^. trypsin solution, maintained at 37° C. Hydrogen-ion concentration,bile and buffers were also found to be important factors in excystation of various species ' coccidia (Smetana, 1933; Lotze and Leek, 196O; Doran and Parr, 1962; Nyberg and Hammond, 196^+). Hibbert et al. (1969), studying the effects of pH, buffers, bile and bile acids on the excystation of sporozoites of various Eimeria species including E. tenella , found no excystation when any of the bile acids or bovine or chicken bile was used alone without trypsin. However, they observed excystation of E. bovis and E. ellipsoidalis in bovine bile containing a heavy s^^pension of bacteria and fungi. When

PAGE 22

11 trypsin alone was used only E. bovis oocysts excysted, the other 9 species of Eimeria including E. tenella did not excyst. The precise vjay in v/hich bile acts in excystation of oocysts is not known at present. Doran and Farr (1962) suggested that bile acids may alter the protein or lipoprotein surface of the steidae body in such a way that it is then readily acted upon by pancreatic enzymes and/or may facilitate entrance of enzymes into the intact oocysts through the altered micropyle. Lotze and Leek (I969) found that in adult chickens about ^0-6 minutes may be required for E. tenella oocysts to be carried from the mouth to the large intestine. A permanent opening or micropyle was not observed in the oocyst v^all through vjhich sporozoites might escape. Therefore the release of large numbers of "sporozoites into the digestive tract of chickens requires the wall of the oocyst to be broken, weakened or partially di:solved. Lotze and Leek (I969) observed the walls of many sporulated oocysts expelled through feces to be structurally changed. Development of immunity does not hinder excystation and normal excystation vjill occur in immune and nonimmune chickens under optimal conditions (Horton-Smith et al. . 1963). Liberated sporozoites are fusiform, 10 ;u x 1.5 J^ in diameter, transparent and motile. Each sporozoite has a nucleus, a prominent refract ile globule at the rounded end, and exhibits various types of movement. They rapidly invade

PAGE 23

12 the surface epithelial cells of the cecum and then, penetrate the basement membrane to enter the tunica propria through which they either pass free or within the macrophages, to finally reach the epithelial cells lining the fundus of the Lieberkiihn glands vjhere asexual reproduction by schizogony occurs (Challey and Burns, 1959; Pattlllo, 1959)After entry into a glandular epithelial cell, the sporozoite rounds up and becomes a trophozoite which develops into a 1st generation schizont (2i^ )x in diameter) within 2^-^Q hours. The nucleus of the invaded cell becomes hypertrophied (Levine, 1963) and the parasitized cell bulges out into the cecal lumen. Each schizont forms about 900 raerozoites (Tyzzer, 1929) 2-4 ;ji in length 1-1 ^ , in width. Merozoites after release from the mature 1st generation schizonts enter a nevf host cell by direct penetration and migrate into subepithelial layers of the tissue to develop as 2nd generation schizonts. The ultrastructure of merozoites and the fine structural changes have been described by McLaren and Paget (1968) and McLaren (I969). Growth of the 2nd generation schizonts is rapid and within 2k hours mature schizonts, containing numerous merozoites, can be observed. The large 2nd stage schizonts of E. tenella are found in the epithelial cells v;hich appear to have moved from the epithelial layers into the subepithelial layers, submucosa and even into the muscular layers of the ceca. The maturation and release of large numbers of 2nd

PAGE 24

13 generation raerozoites causes extensive destruction of the epithelial cells and severe hemorrhage occurs into the cecal lumen follov/ed by tissue necrosis and thickening of the cecal ;mll by the ^Ith and 6th day. The 2nd generation merosoites are considerably larger than the 1st, averaging about 16 ;u in length, 2 ^i in v;idth and 200 to 350 in number, many of vjhich enter nev/ host cells and begin the sexual phase of life cycle by developing into either macrogametocytes or microgametocytes. Microgametocytes are smaller both in size and numbers than macrogametocytes and the 2 are found in close proximity v^ithin columnar epithelial cells of the ceca, belov/ the host cell nuclei. From each microgaraetocyte, microgaraetes develop. Each microgamete has 3 flagella and is motile. Young macrogametocytes are large irregularly shaped cells measuring approximately 5-3 ;um x "^ .3 ;-m. Though ["he young macrogametocyte still retains the shape of the merozoite, it can be distinguished from schizont or microgametocytes by the presence .of "v:all forming" or membraneous bodies (Scholtyseck, 1962) under the electron microscope. Later "dark bodies" develop vrhich are thought to correspond to the "plastic granules" described from light microscope investigations (Reich, 1913; Doflein and Reichenovj, 1953; Cheissin, 1958). After fertilization these bodies migrate to the periphery of zygote. The limiting membranes of the zygote then separate from the cell to become the outermost

PAGE 25

1^ membrane of the oocyst vrall. The middle layer of the cyst v/all is developed from the "dark bodies" and the "wall forming bodies" give rise to the inner layer of the oocyst wall. Thus, in E. tenella the oocyst v;all is trilarainate. When the oocyst vjall is complete the oocyst is extruded from the host tissues and is passed to the exterior vjith the feces. The period from the time of infection to the 1st appearance of oocysts is usually 7 days. The oocyst production thus commences on the 7th day following exposure, reaches a peak by the 10th day and rapidly decreases. Oocysts of E. tenella are ovoidal, clear, transparent, with a V7ell defined double outline. The outer layer of the oocyst wall is quinone tanned protein and the inner layer is a lipid coat firmly associated with a protein lamella (Monne and Honig, 195^). The size of the oocysts range from 1^.2 ;i to 31.2 jLi X 9-5 Ai to 2^.8 /i, with a mean of 22.96 (± 2.2) X 19.16 (± 1.69) M (Backer, 1956). The optimum temperature for sporulation of the oocysts is 29 ± 1° C. (Edgar, 1955) and at this temperature sporulation V7ill be completed in 18 hours but at room temperature it takes about 4-8 hours. The sporulated oocysts contain 4 sporocysts each containing 2 sporozoites. Like in other species of Eir.eria meiotic division occurs during sporogony. E. tenella can be cultivated in the developing chick embryo (Long, 1965, 1966, 1971) and in tissue culture cells (Patton, 1965; Bedrnik, I967, 1969; Strout and Ouellette,

PAGE 26

15 1969; Matsuoka et al. , 1969; Doran, 1970), using sporozoites obtained by in vitro excystation. Pathogenesis of E. tenolla Infection Factors affecting pathogenicity of E. tenella include the number of oocysts ingested', the number of host cells destroyed per ingested oocyst, the degree of reinfection and the state of immunity in the host. The severity of the disease depends upon the interplay of these known and other unknown factors and range from an imperceptible reaction to death (Gardiner, 1955)Cecal coccidiosis under field conditions occurs pi'incipally in young chickens but seldom in those less than 10 or 11 days old. The range of age of susceptibility is from 2 weeks .to 15 months. Many of the worst outbreaks occur at the age of 6 to 8 vjeeks (Blester and Schvmrte, 1965). Herrick et. al . (1936) in a study on experimental infection found that the heaviest mortality and greatest decrease in erythrocytes occurred in chicks 1 month old; heavy mortality also occurred in chicks aged 2 vjeeks and 2 months while in older birds (3i ^, 7, 10, and 15 months of age) mortality v^fas lovi or lacking though drop in red cell count ranged from 29;t to ^^6.8;o. Gardiner (1955) employing a dosage of 50,000, 100,000, and 200,000 sporulated oocysts infected young chickens in age groups of 1, 2, 3, ^, 5, and 6 weeks. Those in the ^ week group v:ere severly affected and those in the 2 vreek group v;ere least affected. Those birds which

PAGE 27

16 recover from infection become immune to reinfection with E. tenella . Hov/ever, this is not an absolute immunity. Under conditions of stress, the acquired immunity of older birds may break down causing symptoms of the disease to reappear (Levine, 1963)Levine (19^' 0) in a study of subclinical coccidial infection in pullets at least 8 months old reported the presence of E. tenella in 2'yfo . In general, however, it can be shown that chicks rigidly isolated from infection remain fully and uniformly susceptible throughout their lives and that age per se has no influence on resistance. Under field conditions almost all chickens get early exposure to at least light infection and so nearly all chicks more than a few days old have some degree of resistance . Inherited resistance in some strains of chickens to infection with E. tenella has been reported by Rosenberg (19^1) and Rosenberg and McGibbon (19^8) but in general there is little evidence of any significant variation in susceptibility between different breeds or strains of chickens (Horton-Smith and Long, I963). Jeffers and Wagenbach (I969) reported higher susceptibility and mortality of female chick embryos from widely different genetic sources to E. tenella infection. Edgar and Herrick (19^1-^) produced evidence to sho\^ that the presence of food in the digestive tract of birds at the time of infection reduced the severity of the disease. Holmes et_ al. (1937) suggested

PAGE 28

17 that increased death rate may result in chickens having higher amount of oyster shells in the ration. The number of oocysts resulting from an infection is not a true indication of the degree of infection. Tyzzer (I929) postulated that, theoretically, infection v/ith a single oocyst of E. tenella could give rise to approximately 1,800,000 oocysts in a period of ^ to 5 days. During the course of an infection, hovvever, there are several factors v/hich may cause a reduction in this potential including loss of merozoites, over crowding, and tissue damage vfhich results in a loss of suitable cells. Brackett and Bliznick (1949, 1952) reported that for each oocyst of E. tenella inoculated in light infection approximately 10,000 oocysts are produced, and there is no direct correlation between the size of infective dose and the final degree of ini tion. Hov/ever, Johnson (I927) reported that the severity of cecal coccidiosis depends on the number of sporulated oocysts that the bird receives. Jankiev/icz and Scofield (193^) reported that a dosage of up to I50 sporulated oocysts produced neither symptoms nor mortality; 150 to 500 oocysts produced slight hemorrhage and no mortality; 1,000 to 3,000 oocysts a fairly heavy degree of hemorrhage and moderate mortality and over 5,000 oocysts produced severe hemorrhage and high mortality. The prepatent period in E. tenella infection is 7 days but the patent period varies viith individual infections. Fish (1931) reported

PAGE 29

18 that oocysts were not .present in the droppings of the infected birds after 1? days although Tyzzer et al. (1932) recorded oocyst passage for as long as 19 days postinfection. The greatest numbers of oocysts are discharged in a very short time (Tyzzer e_t al. , 1932), the fev/ remaining being trapped either in the tissues, or in the cecal contents, and irregularly released. Under natural conditions, birds are usually infected repeatedly and thus may pass oocysts for long periods of time. For example, Levine (19^1-0) observed oocysts of E. tenella in the droppings of 9 out of 30 birds which did not show any symptoms of infection. The disease symptoms in cecal coccidiosis are closely related to the course of infection and, in general j the degree of pathogenicity is related to the depth to which the cecal vrall is parasitized. E. tenella penetrates deeply and is very destructive. During the development of the parasites there is a migration of parasitized cells into the subepithelial region where they increase enormously in size. Much tissue is destroyed and sloughing of mucosa occurs at the time of maturation of 2nd generation schizonts as early as the 96th hour after infection and profuse hemorrhage occurs due to mechanical damage to the blood vessels. This bleeding is the most important effect of the parasitism. Mortality is likely to be great when profuse and continuous bleeding occurs from the ^1-th to 7th days post-exposure. Much of the damage may also come from

PAGE 30

19 secondary bacterial infection of the area in v;hich the epithelium is completely destroyed (Briggs, 1968). Hemorrhage is a great stress on the infected chicken and feeding and movement are at a minimum during this period, but consumption of v/ater is increased 2 to 3 times that seen in uninfected birds. In a typical severe infection, bloody droppings vjill occur 96 hours post-exposure and passage of large quantities of blood in the droppings on the 5th and 6th day post-exposure occur. The disease is at its peak on the 7th day post-exposure and ^0% of the mortality occurs vfithin 9 days follovring initial exposure to oocysts. Chickens surviving 9 days follovjing exposure vrill usually recover. A chronic condition, however, may occur as the result of retention of a core of necrotic tissue in the ceca with consequent cecal dysfunction. In the flock as a v;hole, the disease is nearly alvfays of short duration. It is often found that a condition of disease arises only when infection, heavy in relation to the previous experience of the birds, is acquired during a period of 72 hours or less. If infection is picked up more slowly, then the birds become resistant before clinical effects appear. When chickens are raised on deep litter as in most parts of the xMorld, the oocysts are not necessarily destroyed by the heat of fermentation, but due to the unfavorable environment are predominantly unsporulated. Changes in the environment, notably an increase in moisture and/or temperature.

PAGE 31

20 favor a high and rapid rate of sporulation leading to a clinical disease in the flock. Appearance of fresh blood in the droppings and sudden death are of diagnostic value in cecal coccidiosis. Clotting of tlood is prevented during the acute stages by some unknovm factor(s) (Davies e_t al . , 1963) and deficiency of vitamin K increases pathogenicity to E. tenella and E. necatrix (Davies _ejt al . , 1963). Blood drops can be expressed from the vent of dead birds picked up within a few hours. At necropsy, blood-filled ceca and presence of developmental stages of E. tenella can confirm the dia-MOsis of cecal coccidiosis. The mere presence of oocysts ir not indicative of disease since in E. tenella infections, oocysts are not ordinarily seen in an infection sufficiently acute to cause disease and death (Davies et^ al . , 1963). For experimental infections of coccidiosis, knovm numbers of sporulated oocysts are administered orally. However, Davies and Joyner (1962) and Sharma and Reid (l^o2) succeeded in producing cecal infections by introducing E. tenella oocysts subcutaneously, intravenously, intraperitoneally or intramuscularly in chickens. VJhen viable sporozoites vieve introduced by the same routes, light infections were produced. The method of excystation and transfer to the site of infection for chicken coccidia inoculated parenterally is not yet understood. Horton-Smith and Long (I963) confirmed these findings partially in that

PAGE 32

21 although they could obtain oocysts in the coca aft-er intravenous and intramuscular administration of oocysts, they failed to recover oocysts from ceca vrhen chickens v;ere inoculated intraperitoneally or subcutaneously . They assumed that oocysts inoculated into the blood stream v/ould be removed from the circulation by the liver along viith other foreign bodies. They reported the presence of disintegrating oocysts in the liver of chickens inoculated intramuscularly and suggested that the sporocysts and sporozoites might reach the intestine and ceca via the bile duct. Patnaik (1966) reported that when oocysts v/ere placed in Hillipore chambers grafted within muscles, excystati.on took place inside the chamber with the help of enzymes produced by the infiltrating leukocytes. He also found engulfment of sporozoites by macrophages inside the chamber and postulated that they might carry the spoi . to various parts of the body. In all these cases much lighter infections occurred after parenteral inoculation compared vjith the infections occurring after oral inoculation (Horton-Smith and Long, 1963). Pathology of Cecal Coccidiosis Involvement of the ceca rather than of the small intestine is one characteristic of E. tenella infection. However, if the ceca are surgically removed or, in very heavy infections, the terminal portion of the large intestine will be parasitized. The lesions associated with

PAGE 33

22 E. tenella infection in the ceca have been described by Tyzzer (1929), Tyzzer et al. (1932) and Mayhew (1937). The dilated part of the cecum is primarily involved and substantial damage is due to the large numbers of relatively large 2nd .generation schizonts present in the deeper lamina propria of the mucosa. Production of specific toxin has not yet been demonstrated although parenteral administration of extracts of oocysts is lethal to rabbits and not to chickens (Burns, 1959)' The pathologic effects of the maturation of schizonts and release of 2nd generation merozoites are hemorrhage and sloughing of the epithelial lining of the cecum, vjhich is sometimes stripped dovm to the base of the subraucosa and its replacement by a core composed of necrotic tissue, coagulated blood, cecal contents, and developmental stages of the parasite, chiefly oocysts. This core is at first adherent to the cecal wall, but later may get detached and lie free within the lumen. An infected bird may pass this core or a blood clot in the droppings. The cecum of recovering birds may regain its gross appearance but remain slightly thickened. In lighter infections, recovery is often complete and rapid, but in heavier infections, recovery is slow and the mucosa often shovi only incomplete regeneration. The continuous hemorrhage from the ^th to 7th day postexposure results in profound anemia vrhich is often the cause of death. The exposed skin and mucous membranes

PAGE 34

23 become pallid. Erythrocyte counts and hematocrit decrease to about 50fo of thG normal on the 5th and 6th day after infection vjith 50,000 E. tenella oocysts, and the values return to normal in about 8 days (Natt and Herrick, 1955) • Natt (1959) observed lymphocytopenia and heterophilia on the 5th day and an eosinophil.ia on the 10th day following infection v/ith E. tenella . There were no significant changes in the monocyte and basophil numbers during the course of infection. A marked leucocytosis began on the 7th day post-infection and persisted through the recovery phase of the disease. Pratt (19^0, 19^1) observed an increase in blood sugar during the acute stages of the disease v/ith a decrease in the muscle glycogen. V/axlov (19^1) also found a rise in blood sugar on the 5th day post-infection and a rise in blood chlorides on the 6th and 7th days post-infection v/ith accompanying reduction in muscle chloride. However, Freeman (1970) found no hyperglycemia or change in hepatic glycogen, but he noted a significant reduction in the plasma lactate concentration on the 1st and 2nd days after exposure 3Jid a rise in cardiac glycogen on the 5th day post-infection. According to Daugherty and Herrick (1952), during the acute stages of the infection, a substance produced in the cecum reduced the ability of the brain of the chicken to utilize glucose but not hexose diphosphate. Thus, in cecal coccldiosis a severe interference V7ith normal phosphorylative ciarbohydrate utilization may occur. Challey (l';62) noted an

PAGE 35

2if increase in adrenal ascorbic acid and adrenal corticosterone concentrations during the acute hemorrhagic phase of the infection. Bertke (1963) found renal clearance of uric acid in chickens infected vfith E. tenella greatest at 2 to ^ days after infection. This studysuggested that death is not due to cecal tissue destruction per se nor is it entirely due to cecal bleeding, but to the failure to recover from an initial shock resulting from the development of large numbers of endogenous stages. It is also reported by Johnson and Reid (1970) that in some cases the gross lesions in the ceca in live birds will be more severe than those in dead birds. Husajev and Surkova (1970) studied the nitrogen metabolism of chickens infected v/ith E. tenella and noted that the total and protein nitrogen decrease within the categories of all ages, on the 3rd and especially on the 5th day and this coincides with the period of development of endogenous stages '-^ the parasite. They assume that these disorders are associated with many factors, such as disorders of fermentation and suction and influence of metabolic product of the parasite. They concluded that on the basis of protein metabolism in the liver, deeper pathological changes occur in young chickens than in older birds. Early work by Levine and Herrick (195^, 1957) showed that the voluntary muscles in infected birds are unable to do more than 50;^ of the work done by muscles of

PAGE 36

25 uninfected birds when stimulated via the nerves.Hov/ever, Freeman (1970) found that v;hen the muscles are directly stimulated they are able to do i;ork and speculated that an impairment of nervous conduction ab the neuro-muscular junction occurred in coccidiosis. As for cellular responses, Pierce _et_ aJ. (I962) shovred that during the primary infection with E. tenella . heterophilpolymorphonuclear cells infiltrate into the submucosa in increasing numbers especially on the ^1-th or 5th day post-exposure when the 2nd generation schizonts are developing and maturing. Lesion scoring has been frequently used to compare quantitatively the extent of gross lesions and pathology. Herrick e_t al^. (19^!'2) first described a method of scoring E. tenella lesions using a to k+ scoring system. This scoring system has been followed by many vjorkers (Ripsom and Herrick, 19^5; Gardiner and Farr, 195^; Guckler, 1957; Bankowski et al. . 1959; Horton-Smith et al. , I96I; Lynch, I96I; Britton et al. , 196'!; Turk and Stephens, 1967; Dunkley, I968). A modification of this was also used by Guckler et al. (1958), Waletzky et. al. (19^9-1950), Ball (1959), Boney (19^8), Farr and VJehr (19-!'5), Levine and Barber (19'-l-7), Wale 1 2 ky and Hughes (19'4-9-1950) . Horton-Smith et al. (1961) used a system for macroscopic grading of lesions and correlated this by microscopic grading of parasitism, depending upon the presence of endogenous stages. Johnson and Reid (1970) used a grading system for gross pathology

PAGE 37

26 supplemented by examination for developmental stages in the cecal contents. Lesion scoring is time consuming. Should other disease conditions such as ulcerative enteritis appear in the pens, more extensive microscopic studies may be required to decide vfhether lesions are induced by coccidiosis. Norcross and Washko (1970) examined intestinal tissues from 73^ cases of clinically diagnosed or suspected cases of coccidiosis histologically and confirmed coccidiosis only in 53.2)'^ of the cases. No ;pecific pathological manifestations could be found in the intestinal tissues of 28.6;^ of the cases. The remaining cases were diagnosed in descending order as ulcerative enteritis, leukosis or Marek's disease, other enteritides, helminthiasis and histomoniasis. These studies stress the necesf^i+of both macroscopic and microscopic examination of tn. intestinal tissues for confirming pathology due to E. tenella infection. Microflora and Hosts In nature, animals live in intimate contact vjith many microorganisms. The microorganisms are thus found either in the immediate environment, on the superficial tissues, or in the gastrointestinal tract of animals. This close association has led, in many cases, to symbiotic relationships betvreen the host animal and the microorganisms. The apparently healthy laboratory and other animals used to study many biological processes carry

PAGE 38

27 their indigenous microflora and these animals are known as "conventional" animals. In contrast, a "germ-free" animal is one from vjhich it is not possible to recover any viable organism. Many laboratories employ tests to detect bacteria, fungi, helminth parasites, PPLO, and certain viruses to determine the germ-free state of the animals (Nev/ton, 1965). The terra "gnotobiote" (Reyniers e_t al . , 19'^9) is also used in referring to the germ-free animal and also animals carrying known species of organisms. A "specific pathogen-free" (SPF) animal is one free of specified microorganisms and parasites known to cause disease (Sabourdy, 1965). The SPF animals are functionally and structurally identical v^-ith their conventional counterparts but their flora and fauna are, to some extent, controlled. The production of germ-fi'^ee or SPF animals is a problem if the particu.i...'-r' animal species have certain congenital infections. Salnonella pullorum in chicks and Toxocara canis in dogs (Reece e_t al. , I968; Griesemeret al. , I963) are two infections transmissible congenitally . Careful selection and isolation of breeding stock free of these infections and prompt elimination of young animals or chicks shovring any congenital infections are necessary to insure absence of such infections in the breeding stock (Reece ejt al . , 1968). These elaborate procedures to establish and maintain germ-free or specific pathogen-free animals for research purposes vrill eliminate the necessity for using experimental animals of unknovjn disease exposures,

PAGE 39

28 age, breeding and most important environmental background. The possible influence of the "macro" and "micro" environment in disease processes can be studied v/^ien a stock of animals derived from a breeding colony is raised under 3 different environments — conventional, SPP and germ-free. A number of workers have attempted to raise germfree animals since Pasteur's speculation in 1885 that the host-microflora relationship is obligate. But the investigations of Schottelius (1899), Cohendy (1912), Cohendy and V/ollman (191^), Kilster (1912), Glimstedt (1936), Balzara (1937a, 1937b), Reyniers (19^^6, 19^9, i960), Gustafsson (19^8) and Miyakawa (195'-^-) proved Pasteur's original assumption v7rong. Novf the germ-free animal has become a very useful tool for studying true homeostasis of the gnotobiotic host, the individual actu". and interactions of microorganisms and the response of the host to these organisms. These interactions have not yet been v/ell defined although a few significant microflorahost relationships have been established. The Germ-Free Chick The embryo of healthy birds is maintained in a germ-free condition within the shell until hatching. This has enabled germ-free chicks, turkeys and other birds to be obtained v:ith relative ease. Gerin-free chickens are very popular as experimental animals and have been

PAGE 40

29 successfully used to study such research problemsas the origin of blood group B agglutinins (Springer et al. , 1959), the growth stimulation of dietary antibiotics (Lev and Forbes, 1959), experiments on tumorigenesis (Reyniers and Sacksteder, 1959), development of parasitic infections (Bradley et al. , 1967), and for a study of the germ-free state per se compared with the conventional animals (Reyniers ejb ad . , 19^9, i960). The germ-free chickens are less clean than conventional chicks due to the high humidity in the imits and also by the frequent occurrence of anal blockage and loose nature of the feces of the germ-free chickens. The morphology and function of the gastrointesti]ial tract are altered in the absence of viable flora in the germfree animals (Reyniers, 196O). The intestinal mucosa had less lymphatic development and less connective ti ? mass in germ-free chickens and the general picture v;as that there were more absorptive elements but fewer and less well developed elements of defense (Reyniers e_t al. . i960). Gordon (i960) founo about 3 times greater numbers of reticulo-endothelial cells in the mucosa and the submucosa of the ileum of young conventional chickens than in germ-free chickens. This difference was also noted for "schollen" or globule leukocytes found within the epithelium and in the number of plasma cells and lymphocytes in the submucosa and lamina propria of the lower

PAGE 41

30 ileum. Hdvfever, the -epithelial cell content vjas greater in germ-free chicks . The amount of lamina propria in the total area studied vias 25-6 ± 0.9fo in germ-free chicks and 36.8 ± U\'fo in conventional chicks. Eyssen and DeSomer (I967) reported that the v^e.ight of the small intestine vras 105/;^ greater in conventional than in germfree chicks. Enlargement of the cecum v;as a notable change in several species of germ-free animals including the rat, mouse, rabbit and guinea pig (Wostmann and Bruckner-Kardoss, 1959). However, cecal distention vms not observed in germ-free chickens and turkeys (Luckey, 1963). The ceca of the germ-free chickens vrere found to be significantly shorter in length than those of chickens with a normal bacterial flora (Hegde e_t al. , 1969). From gross observation, the large intestine and cloaca, of conventional and germ-free chicks v;ere similar. Hov/ever, the relative wet weight of large intestine per 100 grams body weight vias greater in germ-free Leghorn chickens. The feces of germ-free and conventional chicks were grossly similar, but the germ-free chicks v:ere more susceptible to diarrhea. The lymphatic system is poorly developed in germ-free animals. Using the ileo-cecal tonsils of birds as an index of the lymph node development, generally a great difference was found (Reyniers e_b al . , 196O; Gordon, i960) between germ-free and conventional chickens. The ileo-cecal tonsils of conventional chickens v;ere of larger

PAGE 42

31 size, full and more turgid v/hile ileo-cecal tonsils of germ-free birds V7ere flabby, pale and inconspicuous. This difference persisted till 5 months of age and from then on the difference became less apparent. The relative v;eight of the trident at the ileo-cecal valve vrhich contains both the ileo-cecal tonsils v/as consistently and substantially smaller in germ-free chickens than in conventional birds. The concentration of lymphocytes in the ileo-cecal tonsils of the germ-free birds ;7as from 1/5 to 1/2 of that found in birds harboring live bacteria. Thorbecke (1959) found no plasma cells or secondary nodules in the ileo-cecalcolic junction of germ-free chicks at 2, ^l, 8, and 14 v:eeks . They vjere found in conventional chicks of all ages and in germ-free chicks at 6 vfeeks of age. In White V/yandotte Bantam chicks, the bursa of Fabricus of conventional chicks were larger per 100 grams 'Dody weight than that of germ-free chicks. " In germ-free chickens the spleen was of smaller size (Reyniers et_ al . , I960) but the general structure, color and consistency of the peripheral and cut surfaces v:ere identical vjith that seen in conventional groups. Distribution of plasma cells vras similar in both the groups but these cells were less often found in the thymus, ileo-cecal junction and follicles of the bursa of the germ-free chicks (Thorbecke, 1959). The growth of germ-free chickens reared on a sterilized purified diet v/as found to be comparable vrith control chicks reared on

PAGE 43

32 natural commercial diet, but the grovfth rate is reported to be faster in gerra-free chicks (Forbes and Park, 1959; Forbes et al . , 1959). Reyniers et_ al. (I960) found growth and reproduction to be normal but egg production and hatchability poor in germ-free chicks. The red cell morphology, hemoglobin concentration, hematocrit values, and the expressed blood volume vrere identical in germ-free and conventional chickens, but leukocytes were 2 to 5 times more numerous in conventional chickens. Circulating lymphocytes v;ere also high enough to state that the presence of living microorganisms and/or their products had an effect on the numbers of lymphocytes (Luckey, 1963). The low level of antibody containing globulin fractions is one of the characteristics of the gern-free animal (Balish and Phillips, 1966; Thorbecke e^ al. , 1957). The gamma-1 , gamma-2 and beta fractions of the globulin fraction of the blood of germ-free chicks 'vrere also low (Wostmann, 1959) and unlike in conventional chicks, no change in serum gamma globulin occurred in germ-free chicks as they matured. It is not knovrn if the low gamma globulin of the gerra-free chicks is "innate" or produced as a result of an unknown antigenic stimulus. Boggs e_t al. (1967) studying granulocytopoiesis in germ-free mice reported a lower concentration of neutrophils in blood of gerra-free mice but the presence or absence of microorganism did not alter the overall granulocytopoiesis. A more positive oxidation-reduction potential of

PAGE 44

33 cecal contents appears directly related to .the absence of gastrointestinal microflora. Balish and Phillips (I966) and Springer (1968) found the oxidation-reduction potentials of bacteria-free cecal contents of the chicken strongly positive v;hile those of the conventional chicken vjere strongly negative. Balish and Phillips (I966) reported that the pH vms higher in all segments of the gut in germ-free chicks when compared to that seen in conventional chicks. Tlie above studies indicated that the germfree chicks shov; acceptable normal grovrth and reproduction. In addition, porosis or spontaneous tumors are not common in germ-free chicks. They survived x-irradiation better than conventional chicks when the dosage was below 800 r at the rate of 32 r per hour (McLaughlin et_ al . , 1958). No gross physiological abnormality has been reported in germ-free chicks. One condition called "jitters" v:as reported by Gordon et_ al. (1959) in germ-free chicks due to cellular proliferation in the brain. The serum of germ-free animals has been shovm to have a low globulin content and very few anti-bacterial agglutinins, though complement and heteroheraagglutinins are present. This, together with paucity of leukocytes and poor phagocytic response, may make the germ-free animals very susceptible to pathogens, although, surprisingly, germ-free animals were found to be very effective in clearing injected particles or dead bacteria (Luckey, 1963). The pathology of many infectious diseases has been studied in gei'm-free animals.

PAGE 45

3^ Pat hop:eni city of Intestinal Parasites and Parasifcism in Gnotobiotic Hosts •• ' Several investigators have utilized gnotobiotic hosts for the study of certain aspects of the host-parasite relationship. In contrast to the research on axenic (in vitro ) cultivation of parasitic organisms which has been directed tov;ards learning the biochemical and immunochemical characteristics of the organisms, the use of gnotobiotic hosts has been oriented towards the in vivo study of the etiology and pathogenesis of certain diseases. A number of studies on the development and pathogenesis of intestinal protozoa and helminths of human and animal importance have been done in gnotobiotic hosts. These studies have shown the relationship between the host, the parasite and the host's intestinal microflora (chiefly bacteria and fungi). Phillips et_ al. (1955) showed that Entamoeba histolytica can produce pathological lesions in guinea pigs only vjhen species of bacteria such as Escherichia coli and Aerobacter aerogenes are present. However, another parasitic protozoan, Pentatrichomonas ( Trichomonas ) hominis . developed in large numbers in germ-free guinea pigs (Phillips, 1962). Pentatrichomonas ( Trichomonas ) vaginalis , iihen subcutaneously injected into germ-free guinea pigs, produced large lesions, but similar administration in conventional guinea pigs resulted in disappearance of the protozoans in a few days (Ne^^fton et al,. , I960). Experimental infection of gnotobiotic mice with Nematospiroides dubius and Nippostrongylus brasiliensis

PAGE 46

35 have been produced and studied (Nev/ton et_ al . , 1959; Westcott, 1971). In general, more parasites developed, infections were of longer duration, and more helminth eggs were produced in the conventional than in germ-free hosts. Eosinophilia was marked in germ-free mice folloviing nematode infection, but no eosinophilia was seen in conventional mice. Nodule development in the intestinal vjall was seen • in both types of hosts, however, the nodules disappeared from conventional hosts rapidly but persisted up to 60 days in germ-free mice. VJeinstein ejt al,. (1962) reported that the larvae of N. clubius vrill not develop to the infective stage in feces from germ-free mice as they normally do in feces of conventional mice. At least in this car; , the intestinal microflora contributed to the prolonged survival and egg production of the helminth species. Another interesting study by Newton _et al. (1959) revealed that the mouse helminths N. dubius and Hymenolepis nana , which do not develop to maturity in the conventional guinea pigs, can do so in the germ-free animal. Johnson et al_. (196?) and Rohovsky and Griesemcr (1967) found feline infectious enteritis in the germ-free cat is a mild, nonfatal disease vjith symptoms of leukopenia, thymic atrophy and lymphoid depletion, but v?ithout morphologic intestinal lesions and clinical signs. In SPP cats, clinical signs, ultrastructural alteration of the intestinal mucosa and reduced enzyme activity were noted (Johnson et_ al . , I967; Fovjler and Rohovsky, 1970).

PAGE 47

36 Using bacteria-free turkeys, Doll and Franker(1963) and Franker and Doll (1964) indicated that host's flora may affect the course of infection with the nematode H. p:allinarum or the protozoan H. melea,p:ridis . Subsequently Bradley e_t al. (1964) and Bradley and Reid (I966) demonstrated a dual etiology involving a protozoan (H. meleagridis ) and a single species of bacteria (E. coli . G. perfringens or B. subtlis ) for infectious enterohepatitis in turkeys. Springer e_t al. (1970) reported that the bacterial requirements for producing infectious enterohepatitis in bacteria-free chickens were different from those for the disease in bacteria-free turkeys. They attributed the role of bacteria in the pathogenesis of infectious enterohepatitis as to make the cecal environment suitable for the survival of H. gall inar urn since histomonads have been found to survive in bacteria-free hosts. An enhancement of virulence cannot be overruled lu this infection as in the case of human amebiasis. In his early studies on the araeba-bacteria relationship Phillips (1964) stated that it vjas almost certain that the bacterial flora acted by "providing a suitable environment/ physical and chemical, for excystation and establishment of lumen infection until such time as the ameba enter the tissue." Recently, Phillips and Gorstein (I966) have demonstrated that various bacterial species alter the virulence of E. histolytica grown in araeba-trypanosome cultures, as

PAGE 48

37 measured by subsequent inoculation into animals. V/ittner and Rosenbaum (1970) studying the role of bacteria in modifying the virulence of E. histolytica found that the increased virulence is associated only vjith contact of ameba with live bacteria and speculated transfer of an episome-like virulence factor from bacteria to the protozoans. Reid and Botero (1967) reported the growth of the cestode, Raillietina cesticillus . in bacteria-free chickens and concluded that no contribution to tho establishment of the tapeworn or interference from the normal bacterial flora of the digestive tract occurs. Johnson (1971) reported tho grov.'th and development of Ascaridia galli in gnotobiotic chickens and the data indicate an inhibit;-of development of the nematode in the bacteria-free chickens . On the other hand, -Balish and Phillips (I966) reported that oral challenge v/ith Candida albicans resulted in crop infection in all bacteria-free chicks but no infection occurred in conventional chicks. Layton and Simkins (I971), in their studies with f''ycoplasma ^alli septicum . found higher mortality (Sjfo) in germ-free chicks than in conventional chicks (38/0. In. gnotobiotic svrine, Kohler and Gross (I969, 1971) have described diarrheagenic effects due to heat-stable filtrates of broth cultures and whole cell lysates of Escherichia coli and Meyer et al. (1964, 1967, 1971) described a polyserositis-like syndrome

PAGE 49

38 due to E. coll in gerin-free pigs. These studies -all indicate that normal flora has either a beneficial or antagonistic action on many of these pathogenic organisms. Another intriguing role for the associated microflora in the host-parasite relationship, perhaps in determining host specificity, was the resistance of the conventional guinea pigs to Trypanosoma cruzi . Hov^ever, a majority of the germ-free guinea pigs harbored the trypanasomes in their blood following intracecal inoculation (Phillips and Wolfe, 1959). In contrast, the bacterial flora had no role in the establishment and pathogenesis of E. brunetti in the chicken intestine (Hegde et al. , I969). Clark et_ al. (I962) also found very little difference in the pathogenicity of E. tenella in conventional and bacteria-free chickens although there V7as a delay of 12 to 15 hours in the appearance of the 2nd generation merozoites in the feces of gnotobiotic hosts. In a more recent study, Visco and Burns (I966, quoted by Hegde et al . , I969) reported no mortality in ^1 gnotobiotic chickens infected vjith E, tenella as compared to 77/3 mortality in infected conventional chickens. They concluded that a close relationship exists betvreen the host flora and the protozoan in the production of cecal coccidiosis syndrome. Kemp e_t al. (197I) reported a delayed development of endogenous stages of E. tenella in germ-free chicks, especially 2nd generation schizonts, gametocytes and oocysts. There v/as a striking lack of

PAGE 50

39 reticulo-endothelial cells in the lamina propria and submucosa and substantially lov; numbers of mononuclear inflammatory cells. Thus, the effects of the bacterial flora on the pathogenicity of this species remain unresolved.

PAGE 51

KORIiAL MICROBIAL FLORA OP CHICKENS The importance of intestinal microflora to the welfare of the host has been recognized early in the history of microbiology. As a result, the nature of the intestinal microflora of many animal species is vjell documented in the literature (Smith and Crabb, I96I; Willingale and Briggs, 1955; Dubos and Schaedler, 1962; Smith and Jones, 1963; Savage and Dubos, 1968; Ogata and Morishita, I969; Savage ejb al, , I968, 1970; Rail et al . , 1970). In avian species, the microflora studies have been limited to turkeys and chickens. Cook et_ al. (195'-0 studied the effects of antibiotics on the intestinal microflora of turkey poults, Naqi et. al. (1970a, 1970b) studied the intestinal microflora of normal healthy turkeys from 1 day to 8 vreeks of age and also in those infected vrith "bluecorab." These studies indicated that in turkey poults shortly after hatching, the intestinal tract is invaded by several species of bacteria.The microorganisms then multiply rapidly reaching high numbers v:ithin the first 2^1to ^8 hours of life. Findings have been similar in other animal species (Lev and Briggs, 1956; Dubos et. al . , 1965; Smith, 1965b). A number of workers have studied the 40

PAGE 52

^1 normal bacterial flora of conventional chickens (Johansson ejb al., 19^8; Shapiro and Sarles, 19^9; Lev and .Briggs, 1956; Lgv et al . , 1957; Huhtanen and Pensack, 1965; Smith, 1965a; Timms, I968; Barnes and Impey, I968). Factors such as age, alimentary tract structure and function, diet, feeding habits and environmental factors have been shown to influence the bacterial flora of the normal gut (Johansson et al. , 19^8; Smith, 196I; Smith, 1965a, 1965b; Smith and Crabb, 196I). All these studies indicated that the numbers of bacteria of all groups were found to be highest in the cecal contents and progressively lov;er numbers in the posterior large and anterior small intestine, respectively. The organisms constituting the major part of the flora v;ere E. coli . enterococci ( Streptococcu.fecalis ) . Lactobacillus sp. , Bacteroides sp. and C. per f ringe d." . The absence of Bacteroides sp. in the small intestine, the preponderance of Bacteroides sp. and Lacto bacillus sp. in the ceca, and the low levels of C. per fringens in all sites v:ere of particular interest to the investigators. Shapiro and Sarles (19^9) found the count of aerobic and anaerobic bacteria to be similar in chickens of different ages. On the contrary, Huhtanen and Pensack (1965) found a preponderance of anaerobes after 2 weeks of age. Their results also indicated that in day-old unfed chicks the flora consisted mainly of S. fecalis. These enterococci gradually disappeared from the duodenum after 6 days of age. The cecum also showed an initially high

PAGE 53

^2 count of enterococci and aerobic bacteria. These were replaced by anaerobic types at around 1^ days of age. The normal bacterial flora has been reported to influence the host-specificity of some parasites (Newton ejb al.. , 1959). Another interesting phenomenon is the decrease and/or increase in the population of some members of the flora during certain pathological conditions. Balish and Phillips (I966) reported that in C. albicans infection of the crop, the count of enterococci v;as increased. Naqi et_ al. ( 197Cq) reported significant differences in the intestinal microflora in turkeys inoculated vjith an infectious enteritis ("bluecomb") agent and uninfected control turkeys. The changes were characterized by a rise in total microbial count of the entire intes; ' a significant increase in number of conforms, lactose nonfermenters and Clostridia. Lactobacillus sp. decreased with severe infectious enteritis but increased when the disease was mild. Microflora changes similar to these findings have been observed by Smith and Jones (I963, 1967) and Ogata and Morishlta (1969) in pigs inoculated experimentally v/ith an enteric pathogen. Johansson and, Sarles (19^f-8) noted that in E. tenella infection, a stimulation of the growth of C. perfringens occurred with concurrent decrease of Lactobacillus sp. and a rise in blood glucose level during the 5th to 7th day post-infection. This may be related to an iiiterf erence in glucose metabolism

PAGE 54

^3 and a role for the flora in the pathogenesis of cecal coccldiosis. Thus, in cecal coccidiosis the problem to be studied is vrhether or not the bacterial flora present in the intestine aid or hinder the initiation of the disease and subsequent development of pathological changes. A particular species or a combination of species may (or may not) help excystation of oocysts, subsequent liberation and survival of sporozoites and invasion of cecal epithelium, development of schizonts and/or gametocytes and thereby contribute to the tissue damage.

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MATERIALS AND METHODS I. Production of Gnot obi otic Chickens Gnotobiotic (bacteria-, fungiand PPLO-free) chickens vjere raised in flexible plastic film isolators follov;ing the method of Bradley et al. (1967). All isolators, accessories and supplies were obtained from the same source.^ The methods employed in sterilization, maintenance, and operation of gnotobiotic environment chambers and equipment, the sterilization of feed supplies and the scheme used for determining the microbiological status were the same as those described by Bradley et al. (196?) • Day-old or 19-day-old embryonated White Leghorn chicken eggs vjere obtained from a commercial hatchery*^ free of Salmonella and Mycoplasma infection and incubated at the laboratory. Prior to introduction into the isolator chambers, all eggs were candled at least tv:ice to insure the viability of the embryos. The surface of the egg shells was sterilized by immersing the eggs (packed in a tubular nylon net) for 8 minutes in a 2% solution of '•G. F. Supply Division, ^31 North Quentin Road, Palatine, Illinois 6006?. p Florida State Hatcheries, Gainesville, Florida 32601. 44

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^5 mercuric chloride held at 37° C. The eggs v;ere then .drawn into the presterilized isolator by means of an egg chute and placed in a plastic tray containing a cotton towel. After removal of the egg chute and sealing of the entry port, the entire isolator vjas placed in a room held at 37° C and 80-85>^ relative humidity for hatching. After hatching, the egg shells v/ere removed and the chickens transferred to a plastic vjire-f loored basket inside the isolator. Sterile feed and v;ater vrere provided ad libitum . II, Production of SPF Chickens Chickens hatching from fertile eggs obtained from the same source as that from which eggs v;ere obtained for production of gnotobiotic chickens v;ere immediately transferred to modified Horsf all-Bauer units. Altogether 10 such units were kept in a room adjoining those in v/hich the plastic film isolators v;ere kept for the production of gnotobiotic chickens. Air entering the Horsf all-Bauer units vms sterilized by passage through sterilized fiberglass filter media. Sterile water was supplied in gallonsized bottles attached to the inlet tube of the unit and the level controlled by gravity flow. Feed consisted of chicken starter mash free of any antibiotics or added chemicals and v/as of composition meeting National Research Council (NRG) standards. The feed was pasteurized in a hot air oven at 150° C for at least 60 minutes and vjas supplied to the chickens in a metal self-feeder inside

PAGE 57

46 each unit. Temperature was controlled eleefcrically and ventilation vias fan-forced, negative pressure. Before and after each use, the units vfere cleaned and scrubbed vfith hot detergent solution and steara sterilized. As far as possible, the units viere opened only 3 times during an experiment — to enter nevjly-hatched chicks, for exposure of the chicks to E. tenella oocysts, and to remove dead birds. Periodically, clinical laboratory tests were conducted to check the pathogen-free nature of the birds. All experimental chickens raised in these units were monitored by standard laboratory methods for the following specific pathogens: 1. the 9 species of Eiraeria causing coccidiosic in chickens ; 2. the common intestinal helminths of chickens (Ascaridia, Heterakis . and Baillietina species); 3. Salmonella and Pasteurella species; and 4. H. meleagridis . III. Production of Conventional Chickens Newly-hatched chickens viere transferred from the incubator and reared in electrically-heated Lattery brooders. Unsterilized chicken starter mash, with no antibiotics or added chemicals, satisfying NRG requirements, and water vrere made available ad libitum . Before and after each use the pens were cleaned, scrubbed with hot detergent solution, and steara sterilized.

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47 IV. Source of Eimerj-a tenella The same strain of E. tenella isolated from a natural case of cecal coccidiosis v;as used throughout this series of experiments. Oocysts were produced according to need in disease-free 3-v^eek-old conventional chickens. Fecal material from donor chickens was collected at 7-9 days after inoculation v:ith a sublethal dose of oocysts and the fecal debris and other gross particles removed by sieving through 30 and 80 mesh sieves. The oocysts viere then sedimented by centrifugation and allov?ed to sporulate in 2fo potassium dichromate solution at room temperature and after sporulation were stored at 5° G. Immediately before use in experimental trials, all oocysts viere surface-sterilized vrith a 0.5^ solution of peracetic acid (Doll et al. , 1963). Sterility v/as tested using standard bacteriological and mycological procedures. Approximately 100,000 sporulated oocysts were administered orally for experimental infection of chickens, using a small syringe with an attached cannula to insure deposition into the crop. V. PatholOFcical Examinations Chickens inoculated with oocysts v;ere kept under close observation. Bleeding or any other clinical signs were noted. Blood samples for packed cell volume determination and serum, analysis were taken both before

PAGE 59

k8 inoculation and on the 7th day after exposure. Bacterial isolates vrere made on both the control and experimental chickens at necropsy, according to the bacteriological procedure described belov;. Chickens dying after exposure to oocysts and all those surviving on the 7th day after exposure vrere necropsied, examined for gross lesions of cecal coccidiosis and the cecal contents examined for various developmental stages of E. tenella . The infection produced in each inoculated group was compared and graded for macroscopic lesions. For histopathological examination, cecal tissues showing gross lesions and cecal tissues from comparable sites from chickens showing no gross lesions vjere fixed in neutral 10/^ formalin and sectioned at 5 or 10 ;ji thickness and stained v/ith' hematoxylin-eosin. The system of macroscopic grading of lesions and microscopic grading of parasitism was modeled after Horton-Smith et al. (I96I) as follows; Scheme of Grading of Lesions and Parasitism Extent of Macroscopic Microscopic Lesions and Grading of Grading of Parasitism Lesions Parasitism No detectable No coccidial stages lesions found even on careful search Small number Small number of of lesions gametocytes found by careful search (5 or less)

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^9 Extent of Lesions' and Parasitism ++ +++ ++++ Macroscopic Grading of Lesions Moderate number of lesions vjith some hemorrhage Numerous lesions and hemorrhage Numerous lesions with severe hemorrhage and cecal enlargement Microscopic Grading of Parasitism Small number of 2nd generation schizonts and/or gametocytes in scattered groups with some associated tissue damage Numerous vjidely distributed gametocytes in localized foci with appreciable tissue damage Numerous schizonts and/or gametocytes v/ith widespread tissue damage VI. Bacteriological Procedures A. Determination of microbial flora of the cecum in conventional chickens The development of cecal microbial flora in diseasefree young conventional chicks and those inoculated with 100,000 sporulated oocysts was studied as follows. Newlyhatched White Leghorn chicks were raised on standard electrically-heated battery brooders and fed unsterilized feed and water ad libitum . Four uninfected control and ^ infected chickens were necropsied at various age intervals: day-old (control only), 6-day-old, 1, 2, 3, ^ and 5 weeks of age. Inoculation with oocysts was adjusted so that the day of necropsy v;ould be 7 days after exposure. At necropsy, the viscera vms exposed and the cecal pouches together vfith 2 inches of anterior small intestine and

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50 posterior large intestine were transferred to a sterile petri dish. Using aseptic procedures, the cecal pouches were opened, the sterile tip (0.0025 crn^) of an inoculating needle was inserted into the cecal contents, withdrawn, and the material on the loop streaked on the surface of each of the various solid media used in a standard petri dish or inoculated into a tube of broth or semisolid medi In case of dry cecal contents or a formed cecal core, a drop of sterile pH 7-0, 0.06? M phosphate buffer was used to soften the material prior to insertion of the sampling loop. Aerobes v/ere enumerated on Difco^^ brain heart infusion agar; enterococci in Difco^ azide-dextrose broth supplemented with 10 p. p.m. methylene blue and 1 . 5fo agar; and conforms on MacGonkey agar. The medium used 'for determining the anaerobic populations was Difco^ brain heart infusion agar supplemented with O.lfo cynteinehydrochloride and 0.1/^ bovine serum. C. D srfringens xvas enumera'ed on Difco^ sulphite polymyxin sulphadiazine agar. For fungus isolation, Difco^ Sabouraud dextrose agar and R Difco Pagano-Levine agar with antibiotics were used; for protozoan isolation,Difco^ Balarauth medium was used. Time of incubation vjas 18 hours for coliforms, 2^ hours for Clostridia and ^8 hours for all other organisms. After incubation, the colonies from each of the 4 uninfected controls and the ^ infected ceca were counted and mean numbers calculated. ^Difco Laboratories, Detroit, Michigan 48232

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51 B. Determination of microbial flora of the cecum in germ-free. SPF. and conventional chickens At necropsy, the primary isolation media used for the recovery and identification of bacteria, fungi and PPLO from the ceca of the germ-freo, SPF and conventional chickens are listed in Table I. The criteria listed by Breed e_t al. (1957) were employed for characterization, identification, and verification of bacterial species. Anaero^ ic plates vfere incubated in a Gaspack Anaerobic Jar #60^10 using Gaspack gas generator envelopes for generating hydrogen and carbon dioxide gas. Methylene blue was used as the indicator. VII. Packed Cell Volume Hematocrit values vieve determined by drav;ing the blood into a heparinized microcapillary tube and centrifuging the blood in an International Microcapillary Centrifuge for 5 minutes at 15,000 r.p.m. The packed cell volume (PCV) was directly read using an Internatic.ial o Capillary Reader. VIII. Total Serum Protein Total serum protein was determined by the method of Weichselbaum (19^6) with slight modifications. A standard ^Baltimore Biological Laboratory, Baltimore, Maryland. 2 International Equipment Company, Needham Heights, Massachusetts.

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52 Table I Primary Isolation Media Used for the. Recovery and Identification of Bacteria and Fungi from Ceca of SPF and Conventional Chickens Difco azide-dextrose broth vfith 10 p. p.m. methylene blue and 1 . 5% agar Difco^ blood agar ^^/ith 0.5/^ defibrinated bovine blood DifccP brain heart infusion agar with O.l^o cysteine-Hcl and ^.1% bovine serum Difco^ Brewer's anaerobic agar Difco^ blood agar with 0.5/o blood agar and neomycin Difco^-Columbia broth Difco enterococci presumptive broth p Difco MacConkey agar P Difco Pagano-Levine base v;ith triphenyl tetrazolium chloride and antibiotics P Difco PPLO broth with antibiotics p Difco Sabouraud dextrose agar with antibiotics Difco^ sulphite, polymyxin sulfadiazine agar Difco^ Salmonella-Shigella agar P Difco thioglycollate medium v/ithout dextrose P Difco tomato juice agar special P Difco trypticase soy broth

PAGE 64

53 curve vjas plotted using various dilutions of a 10/"^ bovine serum albumin solution and corresponding optical density in a Turner Spectrophotometer at a wave length of 5^0 nm. In the procedure, 0.1 ml of unknovm serum and 8.0 ml of stable biuret reagent^ v/ere mixed and incubated at 37° C for A-5 minutes. The optical density was then read at 5^0 nm. The total serum protein was directly calculated from the standard curve and expressed as grams per 100 ml of serum. IX . Serum Electrophoresis Electrophoresis of serum proteins v;as done by Microzone^ Electrophoresis-^ on cellulose-acetate membranes using pH 0.6, 0.075 ionic strength barbital buffer. Using the applicator, 0.50 )xl of the serum sample was applied onto the membrane and electrophoresised at 300 volts for 35 minutes. The membrane then was stained in Ponceau-5 fixative dye solution for « minutes and cleared in 33^ cyclohexanone in absolute alcohol for 1 minute. After drying, the membrane was scanned in a Microzone^ Densitometer and the chart tracings evaluated to obtain the component percentages. ^G. K. Turner Associates, Palo Alto, California. ^Hycel, Inc., Houston, Texas. ^Beclcman Instruments, Inc., Fullerton, California. ^Ibid.

PAGE 65

5^ X. Exposure to Bacteria and. Fuiija:! Single species or combinations of bacteria or fungi to be used along with E. tenella for experimental exposure vjere selected on the basis of microbial isolates from ceca of conventional chickens having very severe coccidiosis. Approximately 0.5 ml of 2^ to -48 hour broth culture of the respective organism v/as administered orally using a cannula. In case of fungi other than Candida sp. , 0.5 ml of a heavy spore suspension was administered. C. albicans was grc n in Difco^ Sabouraud dextrose broth at 37*^ G for ^8 hours and 0.5 ml of the broth was administered orally. Bacterial species were administered 2^ hours before E. tenella oocysts were given; fungal suspensions were given ^8 hours prior to oocyst administration.

PAGE 66

RESULTS Development of Microbial Flora in the Cecum of Conventional Chickens Inoculated vrith E. tenella and Uninoculated Controls The predominant aerobic and anaerobic bacterial species in the cecum at different ages of control chicks and of those exposed to E. tenella oocysts are listed in Table II and Table III. In day-old and 2 -day-old conventional chicks, cecal organisms vrere predominantly enterococci (S. f ocalis ) v;ith E. cpli making up most of the remainder. In noninfected chicks at 7 days of age, enterococci were not found to be the predominajit organisms in the ceca, these being re,, by E. coli and Lactoba.cillus sp. In E. tenella infected chickens, the enterococci vjere greatly outnumbered by other species, especially E. coli and Bacteroides sp., but numbers of Lactobacillus sp. vrere greatly reduced as compared to noninfected controls. On the l^l-th day of age in noninfected conventional chicks, E. coli and Lactobacillus sp. vrere predominant with fevr enterococci, and Bacteroides sp. v/ere rare. In the infected chicks, E. coli vras the most predominant species, and Lactobacillus sp. were not detected. 55

PAGE 67

56 At :?A and 28 days of age, E. coll and Lactobacillus sp. predominated in the noninfected group vihlle in the infected group, E. coli , Bacteroides sp. , enterococci and Lactobacillus sp. viere detected. A stimulation of growth of E. coli . Bacteroides sp. and S. fecalis was observed during E. tenella infection v;hile the growth of Lacto bacillus sp. vjas suppressed. At 35 days of age, the same pattern was observed except that in chickens infected v/ith E. tenella , C. perfringens and anaerobic fecal streptococci were also detected. Fungal isolates vjere rare. In 6-day-old noninfected chickens, Cajidida and Mucor species v/ere detected, but in the infected ceca, only Mucor species were seen. At 1 , 3 and 5 weeks of age, Candida species were detected in noninfected groupsand in 2-vjcek-old infected chickens Mucor species vjere detected. In ^-vjeek-old chickens, no fungi vTere detected in either noninfected and infected groups . Bacterial Isolates in 3-''^eek-01d SPP and Conventional Chickens Inoculated vjith E. tenella and Uninoculated Controls Bacterial isolates in 3-week-old SPF and conventional chickens inoculated with E. tenella were more or less similar and are shovm in Tables VIII and IX. C. per fringens ivas isolated from infected and noninfected SPF chickens and from infected conventional chickens more frequently than from noninfected conventional chickens. A

PAGE 68

51 stimulation of grov/tH of C. perfrin.g:ens was noted both in infected SPP and conventional chickens (TableX). In noninfected conventional chickens, C. oerfringens v/as isolated in only 2 trials vrhile in infected chickens this species was isolated in all the trials. The number of colonies of C. T3erfrin,q:ens developing from a standard inoculum was higher for SPF chickens than for conventional chickens. Occurrence of fungal species v^ras also frequent in SPP chickens, especially in the noninfected ones. Candida species were isolated from noninfected controls in h of the 8 trials, while Candida species v;ere isolated from only 1 of the 8 trials in conventional chickens (Tables VIII and IX). Ki^cor species were infrequent and were isolated only in 1 trial from a noninfected conventional chicken inoculated with E. coli . _C. perfringens and S. fecalis. Pathology due to E. tenella in bacteria-, fungiand PPLO-free chickens is reported in Table IV. Clinical symptoms like bleeding, anorexia, vreakness, and drooping were not noted in any of these chickens. No mortality occurred in 32 of these chickens raised bacteria-, fungiand PPLO-free. The macroscopic grading of lesions at necropsy in all these cases was negative since there was no visible thickening, hemorrhage, core formation, or sloughing of the mucosa. Cecal enlargement was also not noted. The appearance of ceca, liver, kidneys, small and large intestine, bursa of Pabricus, spleen, heart, muscles,

PAGE 69

50 and lungs were normal as compared to the viscera from nonlnfected controls raised bacteria-, fungi-, PPLO-free and also those raised specific pathogen-free and conventionally. Histopathologically, there v/as no tissue damage, hemorrhage, sloughing or thickening of the mucosa evident on microscopic examination. However, E. tenella appeared to survive and undergo some development since endogenous stages were seen in the epithelial cells of the mucosa, especially of the gland fundi. These endogenous stages were identical to immature schizonts and early gametocytes. No large 2nd generation schizonts containing mature raerozoites were seen. Cecal coccidiosis as described by Tyzzer (I929) and Tyzzer et al. (1932) was not seen in these chickens when exposed experimentally to a standard inoculum of E. tenella oocysts. There v^as no decrease in hematocrit values in bacteria-, fungiand PPLO-free chickens exposed to E. tenella (Table XI). On the contrary, hematocrit values increased from a mean pre-infection volume of 27 to 28 on the 7th day post-infection. In chickens harboring only C. albicans , exposure with E. tenella caused mild lesions of thickening but no profound bleeding or sloughing (Table V). Histologically, the tissue damage was negligible but endogenous stages were seen, especially immature schizonts. Denudation of mucosa was minimal. Hematocrit values showed an increase of 1% in these chickens on the 7th day post-infection.

PAGE 70

59 In chickens harboring i'ucor species, moderate numbers of lesions vjith some hemorrhage and thickening v/as noted macroscopically . Histologically, there v/as moderate tissue damage and large numbers of endogenous stages including oocysts were seen. In chickens monocontaminated vjith anaerobic fecal streptococci and exposed to E. tenella there vrere no visible lesions or hemorrhage, but histologically, endogenous stages viere evident. Tissue damage, sloughing of mucosa, hemorrhage, and large 2nd generation schizonts were also absent. Hematocrit values increased from a preexposure volume of 17. to a post-exposure volume of 21.5. In chickens monocontaminated v;ith anaerobic fecal streptococci and a suspension of killed E. coli . C. perfringens . S. fecalis . infection v^ith E. tenella did not produce any clinical symptoms, death, visible lesions, hemorrhage or thickening of cecal mucosa. Histologically, no tissue damage, large 2nd generation schizonts, or bleeding was demonstrated, but immature schizonts vrere seen in the epithelial cells of the mucosa. The hematocrit values v/ere 27 at day of inoculation and 31 on the 7th day post-infection. In chickens monocontaminated v;ith either Bacteroides sp. , C. perfrin.crens , E. col 1 . or other colif orms like A. aerogenes . moderate numbers of lesions with hemorrhage v;ere noticed (Table VI). Death due to cecal coccidiosis occurred only in 2 out of 12 chickens monocontaminated v/ith S. fecalis , and no mortality occurred in those monocontaminated

PAGE 71

60 vfith other species of bacteria. In chickens monocontaminated v/ith S. fecal is there ^^^as a tendency for the blood to coagulate more rapidly. Partially coagulated blood in the ceca was characteristically present in these chickens. Hematocrit values also shov/ed a reduction in chickens raonocontaminated v;ith bacteria and infected vjith E. tenella (Table XI). Comparatively, chickens raonocontaminated either with Bacteroides sp. , C. perfrinp:ens or S. fecalis developed moderate to severe pathology when exposed to E. tenella . Chickens monocontarainated X'/ith E. coli or Lacto bacillus sp. developed only mild to moderate lesions, and those raonocontaminated with anaerobic fecal streptococci showed little pathology xvhen exposed to E. tenella . Chickens infected with E. tenella and polycontaminated with 2 or more species of bacteria or fungi showed lesions intermediate between those of raonocontaminated and SPP or conventional chickens (Table VII). Heavy mortality (80/J) and typical macroscopic and microscopic lesions were noted in chickens polycontaminated with C. perfringens and S. fecalis . Death and typical lesions developed even when day-old chickens polycontaminated with Bacteroides sp. , C. perfringens . E. coli and S. fecalis were exposed to E. tenella . Comparatively, pathological manifestations were more severe in chickens polycontaminated either with G. perfringens and S. fecalis . Bacteroides sp. and S. fecalis than in those polycontaminated with C. perfringens and E. coli . Lactobacillus sp. and S. fecalis. Association

PAGE 72

61 of bacteria and fungi favored development of moderate lesions, but did not favor development of greater pathology. Hovrever, the pathology was more severe than that seen either v/ith the bacterial species alone or fungus species alone. Pathological manifestations in E. tenella -inf ected SPP chickens vjere typical of cecal coccidiosis (Table VIII) Clinical symptoms like bleeding, anorexia, droopiness and mortality v^Jere noted. Thirty-three of the total 88 chickens inoculated died, establishing a mortality rate of 38fo. Macroscopic as vfell as microscopic lesions were observed in all infected chickens establishing an infection rate of 100j2. The mean gross lesion score vjas 2.6 and the mean hematocrit value dropped from a pre-exposure volume of 28.3 to 23.7 on 7th day post-exposure. In conventional chickens, clinical symptoms were identical with those seen in SPP chickens (Table IX). Infection rate v;as 100^'^ and mean gross lesion score was 3-1 Twenty-three out of 10^ inoculated chickens died due to cecal coccidiosis registering a mortality rate of 22.1>o. The mean hematocrit value dropped from a pre-exposure volume of 30.3 to 2 0.1 on the 7th day post-exposure. The clinical symptoms, gross lesions in the cecum and the presence of large numbers of 2nd generation schizonts, extensive denudation of the cecal mucosa and hemorrhage on histopathologic examination confirmed the presence of cecal coccidiosis as described by Tyzzer (I929) and Tyzzer

PAGE 73

62 et al. (1932). In cases having a gross lesion score of ++++ or+++, endogenous stages vjere seen extending deep in the muscle layers of the cecal vrall and bacteria could often be seen among the damaged mucosal layers. The total serum protein concentration and the serum protein fractions in infected and noninfected conventional, SPF and bacteria-, fungiand PPLO-free chickens are reported in Table XII. In conventional and SPF chickens infected with E. tenella there vjas a consistent reduction in total serum protein concentration while such reduction vras not seen in bacteria-, fungiand PPLO-free chickens exposed to E. tenella , when compared to the total ser-um protein concentration of noninfected controls.

PAGE 74

DISCUSSION The cecal microflora v/as found to change in chickens 1 to 35 days of age. Also, changes in certain taxonomic groups of bacteria occurred when the chickens were exposed to standard doses of sporulated oocysts of E. tenella . Earlier workers reported changes in the indigenous microflora depending upon the age, feed, and environment of the chickens. Changes in certain groups of bacteria in the ceca have been reported in cases of coccidiosis by Johansson and Sarles (19^8) and in cases of "bluecomb" by Naqi et. al. (1970a). Enterococci (S, fecalis ) was the dominant organism of the ceca of newly-hatched chickens. E. coli was present in small numbers and became dominant only in v;eek-old or older chickens. These results, as well as the occurrence of large numbers of Lactobacillus sp. and E. coli in chickens aged 1^ to 35 days old, and the constant isolation of Bacteroides sp. agree iirith the results obtained by earlier workers in chickens (Huhtanen and Pensack, 1965; Timras, I968), and in turkeys (Naqi et al. . 1970b). Rail et al. (1970) studied the distribution of bacteria in feces of sviine and identified E. coli . other lactose ferraenters, lactose nonf ermenters , Staphylococcus 63

PAGE 75

6k sp., Clostridium sp. , enterococci and Lactobacillus sp. They concluded that distribution of bacterial cells is nonrandom in feces and organisms might occur as discrete microcolonies rather than as individual cells in samples. Jones and Griffiths (196^) reported that in soil, bacteria occur as colonies and Savage et al, (I968) also reported the occurrence of colonies of bacteria on the walls of thn gastrointestinal tract in rodents. Kolacz e_t al. (1970) using conventional dilution techniques noted more variation among samples than among animals for several groups or organisms. Thus, the concept of bacteria occurring in colonies in fecal samples and in the gastrointestinal .tract makes meaningful ecological interpretation of data derived from conventional dilution techniques difficult because such information may constitute absolute density data and not indicate the actual number of colonies present. Gr^^'^^pv £t al. (1966) reported successful isolation of Bacteroide. sp. from SPF sviine after vigorous sv;abbing of the mucosa to elute the entrapped bacteria. Since the dilution technique has such definite limitations, the technique of taking samples using a standard loop (0.0025 cm^ ) vfas used in this study. This technique has been successfully employed by Rail et al. (1970). The very rare occurrence of Clostridium sp. in apparently healthy conventional chickens in this study agrees vjith the findings of Huhtanen and Pensack (1965). This rare occurrence of species like G. perfringens vjhich

PAGE 76

65 though ubiquitous in .nature, has been variously explained. Lev ejt al. (1957) reported that _C. welchii (C. r3rf rin.p:ens ) was present in day-old chicks in "infected" quarters but not in "uninfected" quarters. They established a correlation betvreen the effects of antibiotics on the young bird in the presence of this organism. Huhtanen and Pensack (1965) failed to substantiate this report. Naqi et al . (1970b) found Clostridia lactose nonfermenters and Bac teroides sp. present in turkeys early in life, but the organisms decreased in numbers or even were absent in older turkeys. They believed that such organisms, on account of their pathogenic and invasive properties, are eliminated by the defense mechanisms of the host, as reported by Dubos et^ al. (1965)* This contention seems unlikely since Timms (1968) reported regular isolation of Bacteroides sp. and C. vfelchii (_C. perfringens ) from ceca of chickens 18 days, 7 weeks, and 5 months old. These species v/ere, hovjever, absent in the small intestine. In the present study, C. perfringens v/as isola.ted from SPP chickens frequently. These SPF chickens were healthy and no "infection" was noticed. They were raised on pasteurized feed and v/ater. Also, Bacteroides sp. vjere isolated from these SPF chickens as well as from conventional chickens. Moreover, in agreement v/ith the report by Johansson and Sarles (19^-8), in chickens exposed to E. tenella . a stimulation of growth of G. perfringens occurred

PAGE 77

66 both in SPF and conventional chickens after exposure to E. tenella. In conventional chickens with cecal coccidiosis, C. perfring:ens was regularly isolated, in contrast to nonexposed controls in which this organism was' isolated in only 2 out of 8 trials. In 1 of those trials, the other chicks of the experimental group were deliberately inoculated v;ith this organism. The mechanisms by which the intestinal bacterial population is caused to fluctuate were not ascertained in this study, but factors such as antagonism betvreen bacteria, competition for common nutrients, and changes in pH and oxidation-reduction potential may play an important part (Hentges, I967, I969; Lev et al., 1957; Meynell, 1963; Schaedler et al. , 1965). The present study confirms the observation of Johansson and Sarles (19^8) that growth of C. perfrin/rens in the cecal flora of chicks shows an increase during infection with E. tenella . and growth of Lactobacillus sp. show a reduction. In 7-day-, 1^-day-, 21-day-, 28-dayand 35day-old chickens with cecal coccidiosis, a suppression of growth of Lactobacillus sp. and a stimulation of growth of Bacteroides sp. , C. perfring:ens . and E. coli was noted. The factor(s) responsible for suppression of an anaerobic species during cecal coccidiosis v/hich occurs abundantly in normal, healthy chickens and stimulation of a species which occurs only sparsely in normal chickens is not yet known. It may be that during the prepatent and/or patent period of cecal coccidiosis, factor(s) favoring

PAGE 78

67 grov7th and multiplication of C. perf rin.g^ens may be readilyavailable v/ith a concomitant reduction of those favoring grovrth and multiplication of Lactobacillus sp. A difference in severity of the cecal coccidiosis syndrome v/as noted betvjeen chickens monocontaminated with C. perfring:ens and chickens monocontaminated vjith Lactobacillus sp. In the former, the pathological manifestations were more severe than the latter. Also, more severe pathology developed in chickens monocontaminated either vjith S. fecalis . E. coli or Bacteroides sp. than in those monocontaminated V7ith Lactobacillus sp. when exposed to standard doses of E. tenella oocysts. This indicates that the influence of microbial flora in the pathology of cecal coccidiosis depends upon the species involved. This is much more clearly evident vfhen the pathological manifestation in chickens monocontaminated with anaerobic fecal streptococci and also those monocontaminated vrith C. albicans is compared v^ith that seen in chickens monocontaminated v/ith other species of microorganisms. Little pathology v/as noted in chickens harboring only anaerobic fecal streptococci and Inoculation of heat-killed E. coli , C. perfringens and S. fecalis did not influence the development of symptoms or pathology. Pathology and clinical symptoms vjere negligible in chickens monocontaminated with C. albicans and only mild to moderate lesions developed in chickens monocontaminated with Nucor species. Under natural conditions, fungi seem

PAGE 79

68 to have little influence in the development of cecal coccidiosis. In healthy conventional chickens, fungi occurred only infrequently. This may be due to the antagonism betv/een the fungi and bacteria vjhich is well knoxm in microbiology. Balish and Phillips (1966) found that in the presence of bacteria, C. albicans was able to produce no lesions in the crop of chickens, but in the absence of other microflora, C. albicans produced crop lesions. In noninoculated SPP chickens, G. albicans was more frequent than in noninoculated conventional chickens. Infection with E. tenella did not influence the growth of this fungus and an inhibition of growth was evident since isolation of fungi in chickens having cecal coccidiosis was infrequent, both in SPP and conventional environments. A comparison of the pathology of cecal coccidiosis in SPP, conventional, and polycontaminated chickens raise" in plastic film isolators clearly shows the influence of the environment and microflora in cecal coccidiosis syndrome. The mortality rate from coccidiosis was higher in infected SPP chickens than in infected conventional chickens, The occurrence and the number of colonies of C. perfringens was higher in SPP chickens than in conventional chickens. This was the only substantial difference in the microflora of these two groups. In polycontaminated chickens raised in plastic film isolators (these are then designated as "conventionalized" chickens), the mortality rate was high ^^'^^ ^' Perfrin.Tens and S. fecalis were members • of the

PAGE 80

69 microflora. A possil:)le role for these organisms in causing death in chickens cannot be ruled out. Balish and Phillips (1966) reported vascular and renal invasion by enterococci in chickens and Domermuth and Gross (I969) reported acute septicemia of young and bacterial endocarditis of older birds due to S. fecalis . Diarrhea, lesions of hemorrhagic necrotic enteritis and death have been reported in nevrborn • piglets due to C. perfringens type C infection (Field and Gibson, 1955; Barnes and Moon, 196^; Bergeland, I965). In conventional chickens the lesions and reduction in hematocrit values as a result of E. tenella infection v/ere more noticeable than' in SPP chickens, despite a lower rate of mortality. Johnson and Reid (1970) reported that, in certain cases, lesions will be more severe in live birds than in dead birds during an outbreak of cecal coccidiosis. The f.-'-lure of chicks to recover from the shock due to the large number of developmental stages within the mucosal layers sufficiently early before cecal bleeding occurs may be influencing the death rate (Bertke, I963). The role of bacterial species like C. perfringens and S. fecalis in the initiation of this suspected shock or in the failure of chicks to recover from this shock, if any, is not knovm. Another feature of cecal coccidiosis, unrestricted hemorrhage with no coagulation, vjas seldom seen in chickens harboring only S_. fecalis. Bacteria may have a definite

PAGE 81

70 role in keeping the blood noncoagulated since polycontamination v;ith _C. perfrinp:ens and S. fecal ip or C. perf rinpcens , E. coli . Bpcteroides sp. and S. fecalis in chickens resulted in classical cecal coccidiosis syndrome with hemorrhage beginning about 96 hours post-exposure, and presence of fresh blood and blood-filled cecal contents on the 7th day post-exposure. Mortality also occurred in these groups. The influence of bacteria as a group and of the various taxonomic groups is more evident vj-hen 1 day-old chickens are exposed to E. tenella . Bacteria-, fungi-, and PPLO-free chickens vjhen exposed on the d.ay of hatch did not develop the pathognomonic lesions of coccidiosis due to E. tenella . Mo mortality occurred in these groups. However, when the inoculation v/as preceded by inoculation with single or multiple species of bacteria, lesions developed Mortality and bloody cecal contents were noted only in the group polycontaminated with Bacteroides sp. , E. coli, C. perfrlngens and S, fecalis . Groups monocontaminated with S. fecalis developed mild lesions, but did not show mortality or blood-filled ceca. The ceca characteristically contained only partially clotted blood. Day-old chickens are generally regarded as less susceptible to E. tenella than chickens 2, 3, or ^ weeks old. Rose (1967b), studying the excystation of E. tenella oocysts, observed rapid and greater excystation in chicks

PAGE 82

71 aged 4, 5and 6 v/eeks and less successful excystatlon in 1 dayor 1 week-old chickens. The greater susceptibility of the 4, 5 and 6 v;eek-old chickens was thus ascribed to the successful rapid excystation of the oocysts and liberation of large numbers of sporozoites in the cecal lumen. The lovj susceptibility of day-old or week-old chickens vjas likev/ise ascribed to the less succe sful excystation of oocysts as a consequence of the less povrerful grinding action of the immature gizzard and dearth of sufficient trypsin in the newly-hatched or young chickens. Trypsin, bile, and hydrogen-ion concentration are some of the factors contributing to the excystation (Levine, 19^2; Ikeda, 1955, 1956, I960; Hibbert et al. , 1969). Hibbert et al. (1969) found that oocysts of many species of Ejjmeria, including E. tenella, failed to excyst if bile alone or trypsin alone is present. They also found that oocysts of E. bovis and E. ellipsoidalis readily excyst in bovine bile containing a heavy suspension of bacteria and fungi. The specific action of the bile or the bacteria and fungi in the excystation is not known. A structural alteration of the v;alls of the sporulated oocysts expelled in the feces of exposed chickens was observed by Lotse and Leek (1969). It is possible that enzymes like trypsin can enter readily into the oocysts whose wall is structurally changed. The present study indicated that development of cecal coccidiosis as

PAGE 83

72 a result of rapid and greater rate of excyctation seems to occur in day-old chickens harboring certain bacterial species v/hich are also regularly isolated from ceca of chickens shovring typical lesions of cecal coccidiosis vjhile excystation and development of the disease seems to be less successful in day-old chickens monocontaminated with S. fecalis . In chickens 3 weeks old, less successful and slow excystation seemed to have occurred when only S. fecalis or E. coli or both vjas harbored in the ceca, if the rate and degree of excystation is correlated with the development of the disease. In the nevjly-hatched conventional chicken the only microorganisms predominant v/ere S. fecalis and E. coli . It was also found that the pathological manifestations of coccidiosis vfere greater if C. perfringens was associated with either S. fecalis or E. coli . Whether C. perfringens aids in the excystation of the oocysts is not knovm and has not been studied. Normal excystation and subsequent liberation of sporozoites may not be the sole process which will influence the development of pathological manifestations in E. tenella infection. Horton-Smith e_t al. (1963) reported normal excystation of oocysts and penetration of liberated sporozoites in immune chickens. It is also kno;^nl that much of the pathology in cecal coccidiosis is due to the maturation of enormous numbers of the large 2nd generation schizonts vihich destroy the epithelial cells.

PAGE 84

73 causing sloughing of the mucosa and hemorrhage. The role of bacteria and/or fungi in favoring the rapid development of the endogenous stages, if any, is not known. The present study shows that typical cecal coccidiosis originally described by Tyzzer (I929) and Tyzzer et al. (1932) failed to develop in chickens raised bacteria-, fungiand PPLO-free. No clinical symptoms, mortality or decrease in hematocrit values following exposure V7ith standard doses of sporulated oocysts were observed. Likev/ise, no visible lesions vrere noted in these chickens. Chickens raised simultaneously under SPF and conventional environments and inoculated with the same standard dose of sporulated oocysts exhibited the classical cecal coccidiosis syndrome. There V7as also a marked difference betvjeen bacteriafree and SPF or conventional chickens based on histopathologic findings. Ceca of the SPF and conventional chickexis inoculated with E. tenella oocysts and necropsied on the 7th day post-exposure showed extensive denudation of the mucosa, hemorrhage and large numbers of 2nd generation schizonts. In severe cases, the hemorrhage and tissue damage even reached the muscular layers, and the entire mucosal layer vras seen to be stripped dc-ra. to the base of the submucosa. Large 2nd generation schizonts lying mostly free and a few within their host cells v/ere the predominant endogenous stage seen in such cases. But in the ceca of

PAGE 85

7^ bacteria-, fungiand PPLO-free chickens exposed to E. tenella oocysts and necropsied on 7th day post-exposure, no extensive denudation of mucosa, hemorrhage or large 2nd generation schizonts were seen. The mucosal layer v;as more or less intact and other tissues viere undamaged; no large 2nd generation schizonts were seen. A few endogenous stages identified as immature schizonts were noticed in the epithelial cells of the mucosa, especially those lining the gland fundi. This location of the stages suggests that they may be the 1st generation schizonts developing very slowly. This suggests that in bacteria-, fungiand PPLO-free chickens the degree and rate of development of endogenous stages are markedly inhibited. This inhibition of the development of endogenous -stages, especially the pathogenic and immunogenic 2nd generation schizont may. afford protection to the host and the pathological manifestations are not developed. In fact, the bacteria-, fungiand PPLO-free chickens seem to behave as an immune host. Excystation has occurred in these hosts since endogenous stages are seen in the host cells post-exposure to E. tenella . but their subsequent development seems to have been retarded. Clark e_t al. (I962), in their studies with bacteria-free chickens, observed a 12-15 hour delay in the appearance of 2nd generation merozoites in the feces of bacterla-frec chickens. However, they found no difference in the course of infection in

PAGE 86

75 bacteria-free and conventional chickens. The exact experimental procedures and results are not completely knovm to compare their results and those from the present study. Wagenbach ejb al. (I966), describing a method for sterilizing coccidian oocysts employing Glorox and sulfuric acid-dichromate solution, indicated that they could produce cecal coccidiosis in "gnotobiotic chickens." Since the exact nature of their "gnotobiotic chickens" has not been indicated nor has the description of the experimental procedure and results of their v/ork been given, it is not possible to compare their results with the present work. Their vjork was designed to obtain sterile oocysts for cultivation of the parasite in tissue culture. Nyberg and Knapp (1970a, 1970b), examining the oocysts of E. tenella by means of a scanning electron microscope, reported certain structural alterations of oocysts. Monne and Honig (195^) reported that v/hen oocysts are treated v;ith sulfuric acid prior to sodium hypochlorite, the outer layer of the oocyst wall became wrinkled and apparently elevated away from the inner layer. Employment of such structurally altered oocysts might influence pathogenesis and development of disease processes of coccidiosis. In contrast, the use of 0.5/^ peracetic acid for surface sterilization of invertebrate

PAGE 87

76 eggs and oocysts has been found to be very effective(Doll et al., 1963; Bradley et al. , 196^1-; Hegde et al . , 1969; Springer et al. , I970). Under the light microscope, no damage or changes to the oocyst wall v/ere seen v;hen peracetic acid was used for surface sterilization and therefore it is assumed that peracetic acid is not injurious to the oocyst wall when used according to the method of Doll et al. (1963) and this method was used during the present study. The data obtained in the present study can be compared to those obtained by Visco and Burns (I966, quoted by Hegde et al. , 1969) who observed no mortality in bacteria-free chicks ^,hen exposed to E. tenella oocysts and by Kemp et al. (1971) who reported a delaying of the development of the endogenous stages in germ-free 'chicks. In the present study, there was also a delay in the development of endogenous stages observed and this delay may be beneficial, allowing the host to develop sufficient resistance and thereby inhibit the development of clinical symptoms and other manifestations of the disease. It is also possible that the development of the large 2nd generation schizonts, if delayed or inhibited by the absence of microflora, will lead to minimal development of pathology. Inhibition of development and development of fewer numbers of nematode parasites have been found in germ-free hosts including the chicken (Newton st al., I959 Johnson, 1971). Springer et al. (1970) reported survival

PAGE 88

11 of H. melea,?ridis v/ithout disease production, in gnotobiotic hosts. Confirming the synergistic role of certain bacterial species (especially C. perf ringens ) and H. meleagridis in the proiuction of infectious enterohepatitis in turkeys reported by Bradley e_t al. (196^), Springer et al. (1970) established that the role of bacteria is more vital for the survival of H. gallinarum larvae than H. meleagridis in the pathogenesis of infectious enterohepatitis . In E. tenclla infections, the exact role of the bacterial species is not knovm, but the present study indicates that for the disease syndrome to develop in full, certain bacterial species are essential since no mortality or gross lesions were seen in hosts free' of any detectable organisms. Johnson ejt al. (196?) and Rohovsky and Griesemer (196?) found feline Infectious enteritis in the germ-free cat to be a mild, nonfatal disease consisting of leukopenia, thymic atrophy and lymphoid depletion but vrithout morphologic intestinal lesions. On the contrary, SPF cats developed clinical signs of typical intestinal involvement as seen in frank infectious enteritis. Since the ceca of the chickens contain the major portion of the gastrointestinal flora (Timms, I968), E. tenella may have developed an apparent obligate relationship vrith certain members of this microflora for pathogenesis to commence and this relationship would have influenced the overall host-parasite relationship.

PAGE 89

78 The contribution of indigenous bacterria of. the cecal pouches in producing cecal coccidiosis syndrome vjas first suspected by Ott (1937). Mann (19^7) reported the bacteriology of cecal coccidiosis to be similar to that of "six-day disease" incited by G. perfrinp:ens . enterococci and coliforms, and characterized by occlusion of ceca V7ith a core and hemorrhage. Johansson and Sarles (19^8) also reported a possible involvement of E. coll in the etiology of cecal coccidiosis. Their study and the present study shov;ed that coliforms are present in large numbers throughout the course of cecal coccidiosis. They assumed that the reduced severity . in older chickens is due to low coliform count in the cecum. The reduced severity of cecal coccidiosis" se in day-old chickens may also be due to the same reason.

PAGE 90

SUHMARY AND CONCLUSIONS Typical cecal coccidlosis did not develop in bacteria-, fungiand PPLO-free chickens inoculated v/ith a standard dose of infective oocysts of E. tenella . However, small numbers of E. tenella were able to develop at a slovfer rate in bacteria-, fungiand PPLO-free chickens as compared with the rate seen in conventional chickens. In chickens monocontaminated vjith C. perfrin gens , S. fecalis . E. coll . Bacteroides sp. , Lactobacillus sp. , Huoor sp. or C. albicans ^ exposure to E. tenolla oocysts resulted in raild cecal coccidiosis. Cecal coccidlosis in chickens polycontaminated with bacteria was comparable vfith that seen in conventional or SPP chickens. Higher mortality but lovrer mean gross lesions due to coccidiosis were seen in SPP chickens exposed to E. tenella . compared to that in conventional chickens. Indigenous flora of the ceca of chickens of various ages was not identical. In very young chickens (up to 1 week of age), entsrococci predominatedin the ceca v/hile in older chickens (^ to 5 v;eeks of age), Lactobacillus sp. predominated. Bacteroides sp. , E. coli , Lactobacillus sp. and S. f eca!l.is vjere regularly isolated from conventional chickens at 1, 2, 3, ^ and 5 weeks of age. Isolation of 79

PAGE 91

80 C. perfrln,e;ens v/as infrequent in conventional chickens, but frequent in SPP chickens. Inoculation of chickens vjith E. tenclla resulted in stimulation of growth of C. perfrinQ;ens both in conventional and SPP birds. Also, large numbers of E. coli and Bacteroides sp. , but low numbers of Lactobacillus sp. were seen in the ceca of infected chickens. The data indicate that the indigenous bacteria aid in rapid development of endogenous stages of E. tenella and production of typical cecal coccidiosis.

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APPENDICES

PAGE 93

APPENDIX A

PAGE 94

83

PAGE 95

8^ o m m CD ^ i^ CD b.0 O S-H •H ^ S O o S: rH W O -P O (D •H > o o o s o -d ?H 0) O Kl ft 0) X •H a) o rl a) o w E 1-1 O -d S w n3 -H w iH O •H "d !m -H O 4J O O O CO O PQ +5

PAGE 96

85 CO •H O o CO EH O su p o o -d CO o CD g r-H CD Pi CD -P o\ •H wl o p -d (D CQ o ft W p. f^H O (U o Phi-:1 Cm

PAGE 97

86 e & •H O B
PAGE 98

87 0) § •H O o E-i o

PAGE 99

88 o

PAGE 100

89 EH ri to P^ o -p Cm 03 O iH O K! CO
PAGE 101

90 tt-t

PAGE 102

91 iH a EM O •H (i-i B P< O W O •H'— o ho-p ri O -H Kl Q) Jh rd nj S o 03 y^— ' •H 5h CO o mH Oh ft O W O boo fn cQ ?:! -H a,' O .H W (D J-i 'd 0) ^: o oj t-q^cC fn SO >5 -p 1 -H o aj S -P 05 O CO •H rt •H -H rH cn C« CO -H (U ^ •H
PAGE 103

92 o a o w O •H'— O M4^ S W C .H CO O -H 01 CD Wd ci ^ O CO SL,— •H ?H CO S C^ CM o •H Cm O CO-^ o fafl r; s CQ s o cO O ^H -H Jm fd CO K O CO CD—' CO !h -! o CM w

PAGE 104

93 O o > iH EH O •H Chh £3 P( O U O •H—^ CO s:! -H ct5 O mH W (!) O cC ?:,--' •H ?H CO S C!j (-M O ft o o w O tiO rt — w j:^ O ?:) O -H -H C[5 O n3 Q) rr^: SO I -H o ns O K) •H fj 'rt Go rH to O k5 •H a) 0) o o CO CQ 0) W j Q) ;i; N o w (U -p 01 r-4 • O M (SI o ca r-i o CQ o c^ o I •H O (D O P CQ CU !^ O >3 U -d s CO :3 id r CO fCiJ CQ 4^0 rH CQvO CQ p. •H (D I O X col o Wl O o o o 'd s o CU > -p iH 5:^ O rd >j o rH -H rH -P CO O o rt •H ft 0) O JL4 O CD CQ ^ O ^ CQ O -P •H rt EJ O N rd -H (U ^ o o ^ CQ 4-5 O Q) Pi CQ 4-5 CO CO & E E S M o •H CQ • CD ^ rH CD CD CQ r-i ,Q G) •H fn CQ CD (D CD O to S CO CQ E ^ CX5 CD O >d in •H CD ,C (D £: CQ CQ Si CQ 43 •H .H Si 4J O S N CD fd -H ^ Si ^ O CO o•H CQ s: O bO si CO o S fH43 O Sh CO o S E M Q ^ si rd rH -d •H Si Ecv) 42 o 4J CO ^ Cm O CO d Si p si o d CQ o ft X Si o o

PAGE 105

9^ r-\ (D m "d m -H j bO rd O 03 -d CO CO cti i-l o O 4^ CQ O M CO PQ s O .H -P +:> fe .H O O ;3.H CQ CO mJ OP4 H 2 ^ CO cd E I fiOcO I O Pi fH E O -H -H CVJ W O O CO -P •H O ;h <<-( -H a CO C!3.0 CQ g-5 •H CO CO Q) hf) I O ^, O -H .H !h P-fd o o oj CO O ?M «M (D S s: CQ c5 o t^^--p 5^ rH O CO S -P rH CO O CQ W CQ

PAGE 106

95 •H o o Eh in I ho 03 \ o a ^ e O -H -H Oj CO f^ ft rcJ PL, .H O O C(5 4J hH O ^^ <*-. -H S m O O W to O C •H R5 to Q) ho I o SI! O -H -H o o nJ CO O J^ «M Of-:)--' -p ! -H Jh r-i O rt S 4J o to iH CO O Q)

PAGE 107

96 B I O S Jh S H .H K5 W O Cti -P O f-1 <^ -H w O O m H .H CO •H CD W (D o o nJ C3 O Jh «« Q) S S CQ O O t^ — -p 5h pH O k5 rH o w r-l CO o 03 to .H O c„-P rt Q) O O 3 W (D iH ft4-> -P fi m $:! ,Q «M O E O .H ^ U •H E rH >^ Q)

PAGE 108

97 g -P o M H > O rH ,Q CO EH CO e (D hD CO

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98

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99 EH O •H o (D Q) Sh Ph I QJ hO O ce o CD ft n3 rH 0) -p 5 ho O rH O ^ 4-5 03 PL, w

PAGE 111

100 m •H ctf o u (D o rd ftrH Q) in ^ A-^ O cS sd O O CO -H W ft E Cm o o Q CO

PAGE 112

101 J •H -P o o > (D rH EH W Q) H K) O in Q) O -d P -P S « CO -H a'' CQ u s -Q Cm ^ E O O :3 .H o iH CO ,Q •H E EH £2; ft

PAGE 113

102 CO 0) •H a o u Q) O nd CO «i-i -P O ce -P ?H rH SH O O CO .H CO fi S M •H E
PAGE 114

103 •H -P o o 03 EH w O Jh Q) O nd C/2 «i-i •J O K) 4^ SU rH fl O O E Ch o o o •H fM R p. O U O -H-^ o bo-p s:! O -H W CD O CO ?H— ' SOPM o •H c„ P. O O CQ O fc£) ^^^ CQ j:; o « O -H .H CC U Ti V} CD O CO (D S CO Jh t^ — S C5 >-5 O CO S 4^ CO O C/3 •H .H rH CO ?^ i^! -CO CO 0) iH O +3 ^ 0.1 p.+i -p r; X CO -H CD ?-.

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PAGE 117

106 r.c> (D •H c\3 O U CD O -d PhiH OJ CO <<-i -1^ O CO •P ^ rH S O O C\3 -H W •H o o o O CO !h CO O g: Ph E CO S E 0)
PAGE 118

107 PS o u X iH E-i w •H o3 O Sh Q) O fd CO tM 4J o a -P ^ rH Pi O O Cd -H CO Pi S M •H E «w O O Q O •H Cm e P< O W O -H — O hO-P fl CO iZj -H Ki O .H CO Q) O Cd ?H^-' •H J-i CS o •H Ci-, ft O O CO O W) S'-CO S O S O -H .H CO Pi -d CO 0) o c\5 o j;: ct> p, t^--^ Pt M O ct3 a -P CI? O CO p; bo • H .H iH CO (D p< p! • ct; rS Pi m|m CO ^ CD ft+3 +i j;: X ce .H CD W Ph ^ -P CO P4 Pi ^ Cm ^ BOO 1^ -H ^ Si o p< iH (D CO rO •H e p. ^ E-I a .

PAGE 119

108 ra CD •H CO o u 0) O Ti ftiH 0) W tM -P O CO U rH O O cC •H o o Q CQ rH O O CD C|Cm

PAGE 120

109 •H -P o o X EH CO (D •H CO O !:h 0) o fd P,rH 0) CO Cj_, +3 O td P ^ H rj o O •H ra E Ch o c Q o •H <4H B P, O CO O -H — O W)j3 r! CO ri -H Cd O -H CO 0) ^ nc) cC >S o ce Sh — •H i:.! cti S C5 Ph O •H q-i a. o O CO o fao S'-CO s:; o s O -H -H a5 Jh re) CO CD o ce 0) ;a so f-1 H ft ft ft cti

PAGE 121

110 -d

PAGE 122

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PAGE 123

112 M x: (D

PAGE 124

113 1 -p

PAGE 125

114-

PAGE 126

115 O O 03 EH 1

PAGE 127

116 I -p a o > o o >>.H f^ CO -P ^(^ ^ ^, o -p rt >
PAGE 128

117 I -p 03 O > o Ph 03 -P ^O O S£>-M > 0)

PAGE 129

118 C
PAGE 130

119 •H o o M M -Q

PAGE 131

APPENDIX B

PAGE 132

121 Figure 1. Ceca of 3 vjeek-old specific pathogenfree chicken exposed to Eimeria tenella, 7th day postinoculation. Macroscopic grading of the lesion ++-I-+. Figure 2. Photomicrograph of a transverse section of cecum of 3 vjeek-old specific pathogen-free chicken exposed to Eineria tenella shovring denudation of mucosa, and large 2nd generation schizonts. Hematoxylin-eosin stain. XI25. Microscopic grading of parasitism ++++.

PAGE 133

122 Figure 3. Ceca of 3 vieek-old bacteria-, fungiand PPLO-free chicken exposed to Eimeria tenella , 7th day postinoculation. Macroscopic grading of the lesion 0. Figure ^. Photomicrograph of a transverse section of cecum of 3 vjeek-oid bacteria-, fungi and PPLO-free chicken exposed to Eimeria tenella shovjing a single coccidiura and no denudation of mucosa or other tissue damage. Hematoxylin-eosin stain. X400.

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123 Figure 5. Ceca of 3 vjeck-old bacteria-, fungiand PPLO-free chicken exposed to Eimeria tenella and Clostridium perfringens . Ilacroscopic grading of lesion

PAGE 135

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125 Bedrnik, P. 1967. Development of sexual stages and oocysts from second generation Eineria tenella merozoites in tissue cultures. Folia Parasit. Bedrnik, P. 1969Some results and problems of cultivation of Eineria tenella in tissue cultures. Acta. Vet. 30:31-35. Bergeland, H. S. 1965. Studies of a porcine enterotoxemia caused by Clostridiuni perf rinp:ens type C. Doctoral dissertation, University of Minnesota. Bertke, E. M. 19^3. Renal function during the course of Eineria tenella infection. J. Parasit. ^1-9:937Blester, H. E. and L. H. Schvmrte (eds.). 1965. Diseases of Poultry. Iowa State University Press, Ames, Iowa, p. 828-858. Boggs, D. R., P. A. Chervenick, J. C. Marsh, H. I. Pilgrim, G. E. Cartwright, and M. M. Wintrobe. I967. Granulocytopoiesis in germ-free mice. Proc. Soc. Exp. Biol. Med. 125:325-330. Boney, F. A. 19^w. The efficacy of methyl bromide as a fumigant for the prevention of cecal coccidiosis in chickens. Am. J. Vet. Res. 9:210-21^. Brackett, S. and A. Bliznick. 19^9The effect of small doses of drugs on oocyst production of infections vrith Biraeria " tenellri . Ann. N. Y. Acad. Sci. 52: 595-6T0: Brackett, S. and A. Bliznick. 1952. The reproductive potential of five species of coccidia of the chicken as demonstrated by oocyst production. J. Parasit. 38:133-139. Bradley, R. E. , H. Botero, J. Johnson, and W. 11. Reid. 1967. Gnotobiotic poultry in plastic film isolators and some applications to parasit ologlcal research. Exp. Parasit. 21 :^!-03-^13. Bradley, R. E., J. Johnson, and W. H. Reid. 196^1'. Apparent obligate relationship between Histomonas meieagridis and Escherichia col i in producing disease. J. Parasit. 50:51*

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126 Bradley, R. E. Gnd V/. H. Re id. I966. Hlsto'^on?.s .. mele pp-rldis and several bacteria as agents of infectious enterohepatitis in gnotobiotic turkeys. Exp. Parasit. 19:91-101. Breed, R. S., E. G. D. Murray, and N. R. Smith. 1957Bergey's Manual of Determinative Bacteriology. 7th ed. Williams and Wilkins Co., Baltimore, Maryland, p. 109^. Briggs, B. D. (ed.). 19^8. Coccidiosis of Chickens. Norwich Animal Industry, Inc., Norvjich, New York, p. 1-32. Britton, \-J . 11., C. H. Hill, and C. \1 . Barber. 196^. A mechanism of interaction betvjeen dietary protein levels and coccidiosis in chicks. J. Nutrit. 82: 306-310. Burns, W. C. 1959. The lethal effects of Eimeria tenella extracts on rabbits. J. Parasit. ^5: 38-^6. Challey, J. R. I962. The role of bursa of Fabricius in adrenal response and mortality due to Eimeria tenella infection in the chicken. J. Parasit. ^3:352-357. Challey, J. R. and W. C. Burns. 1959. The invasion of the cecal mucosa by Eimeria tenella sporozoites and their transport by macrophages. J. Prot. 6;2382iH. Cheissir, E. M. 1958. Cytologische untersuchungen vorschiedener stadiem des lebens-zyklus der kaninchen coccidien. I. Eimeria intestinal is . Arch. Protistenk. 102:265-290. Clark, D. T., C. K. Smith, and R. B. Dardas . I962. Pathological and immunological changes in gnotobiotic chickens due to Eimeria tenella . Poultry Sci. ^H:1635-1636. Coates, M. E., J. E. Ford, and G. F. Harrison. I968. Intestinal synthesis of some vitamins of the B complex in chickens. In K. Miyakavra and T. D. Luckey (eds.). Advances in Germ-Free Research and Gnotobiology . The Chemical Rubber Company Press, Cleveland, Ohio, p. 110-111. Cohendy, M. 1912. Experiences sur la vie sans microbes. Ann. Inst. Pasteur. 26:106-137.

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127 Cohendy, M. and E.^ V/ollman. 191^. Presentee par.H. Roux. Bacteriologie. ExpSriences sur la vie sans micrbbes. Elevage aseptl'que de cobayes. Compt. Rend. Acad. Sci. 158:1283-128^. Cook, F. D., R. M. Blakely, H. I. McGregor', and R. \1 . Anderson. 195^The effect of antibiotics on the intestinal microflora of turkey poults. Poultry Sci. 33:38-^1-0. Cuckler, A. C. 1957The effect of nicarbazin on the development of immunity to reinfection with coccidia in poultry. Merck Poultry Nutrition and Health Symposium, St. Louis, Missouri, p. 22. Cuckler, A. C, L. R. Chapin, C. M. Malanga, E. F. Rogers, H. J. Becker, R. L. Clark, N. J. Leanza, A. A. Pessolano, T. Y. Shen, and L. H. Sarett. 1958. Antiparasitic drugs. II. Anticoccidial activity of 4,5-imidazoldicar boxaraide and related compounds. Proc. Soc. Exp. Biol, and Med. 98:167-170. Daugherty, J. W. and C. A. Herrick. 1952. Cecal coccidiosis and carbohydrate metabolism in chickens. J. Parasit. 38:298-304. Davies, S. F. M. and L. P. Joyner. 1962. Infection of the fowl by the parenteral inoculation of ooc.y ' of Eimeria . Nature 19^:996-997. Davies, S. F. M., L. P. Joyner, and S. B. Kendall. 1963 . Coccidiosis. Oliver and Boyd, Edinburgh. Davies, S. F. M. and S. B. Kendall. 1955An experimental assessment of the value of nitrofurazone used continuously as a coccidiostatic drug. Vet. Rec. 67: 867-870. Doflein, P. and E. Reichenow. 1953Lehrbuch der protozoenkunde, Jena: Fischer Verlag. p. 1213. Doll, J. P. and C. K. Franker. 1963Experimental histomoniasis in gnotobiotic turkeys. I. Infection and histopathology of the bacteria-free host. J. Parasit. 49:411-414. Doll, J. P., P. C. Trexler, L. I. Reynolds, and G. R. Bernard. 1963^ The use of peracetic acid to obtain germ-free invertebrate eggs for gnotobiotic studies. Am. Midi. Nat. 69:231-239.

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128 Doraerrauth, C. H. and W. B. Gross. 1969A medium for isolation and tentative identification of fecal streptococci, and their role as avian pathogens. Avian Dis. 13:39^-399Doran, D. J. 1970. Eimeria tenella : oocysts in cell culture. 37:8^-91 Proc, from sporozoites to Helm'. Soc. VJash. Doran, D. J. and M. M. Parr. 1962. Excystatlon of the poultry coccidiura, Eimeria acervulina . J. Prot. 9:15^-161. Doran, D. J. and M. M. Farr. 1965. Susceptibility of 1and 3-day-old chicks to infection v;ith the coccidium, Eimeria acervulina . J. Prot. 12;l60-166. Dubos, R. J. and R. W. Schaedler. 1962. The effect of diet on the fecal bacterial flora of mice and on their resistance to infection. J. Exp. Med. 115: 1161-1172. Dubos, R. J., R. W. Schaedler, R. Gostello, and P. Hoet. 1965. Indigenous and autochthonous flora of the gastrointestinal tract. J. Exp. Med. 122:67-75Dunkley, M. J. W. 1968. Laboratory trials with buquinolate — a new broad-spectrum coccidiostat for poultry. Vet. Rec. 83:30-3^. Edgar, S. A. .1941. The development of the protozoan parasite Eimeria tenella Railliet and Lucet I89I in domestic fov;l. Doctoral dissertation, University of Wisconsin Memorial Library, Madison. Edgar, S. A. 1955 • Sporulation of oocysts at specific temperatures and notes on the prepatent period of several species of avian coccidia. J. Parasit. ^l:21^~216. Edgar, S. A. and C. A. Herrick. 194-4. Feeding habits in relation to the severity of cecal coccidiosis, Eimeria tenella . Poultry Sci. 23:30-35Edgar, S. A. and C. T. Seibold. 1964. A new coccidium of chickens Eimeria mivati sp. n. (Protozoa: Eimeriidae) V7ith details of its life-history. J. Parasit. 50: 193-204. Eyssen, H. and P. DeSomer. I967. Effects of Streptococcus fecalis and a filterable agent on the growth and nutrient absorption in gnotobiotic chicks. Poultry Sci. 46:323-333.

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129 Farr, M. M .• and E. E. Wehr. 19^5Sulf amer?.zine therapy in experimental cecal coccidiosis of chickens. J. Parasit. 31:353-358. Field, H. I. and E. A. Gibson. 1955Studies on piglet mortality. II. Clostridium vielchii infection. Vet. Rec. 67:31-35. Fish, F. F. 1931. Quantitative and statistical analysis of infections viith Eineria tenolla in the chicken. Am. J. Hyg. 1^:560-576. Forbes, M. and J. T. Park. 1959Growth of germ free and conventional chicks: effect of diet, dietary penicillin, and bacterial environment. J. Nutrit. 67: 69-84. Forbes, M. , J. T. Park, and M. Lev. 1959. Role of the intestinal flora in the growth response of chicks to dietary penicillin. Ann. N. Y. Acad. Sci. 78: 321-327. Fowler, E. H. and M. W. Rohovsky. I970. Enzyme histochemistry of the small intestine in germ free and specific pathogen free cats inoculated with feline i^'ectious enteritis (panleukopenia) virus. Am. J. . :.. Res. 31:2055-2060. Franker, C. K. and J.. P. Doll. 1964. Experimental histomoniasis in turkeys. II. Effects of some cecal bacteria on pathogenesis. J. Parasit. 30:636-640. Freeman, B. M. I970. Carbohydrate stores in chickens infected with Eimeria tenella . Parasitology 61: 245-251. Gardiner, J. L. 1955. The severity of cecal coccidiosis infection in chickens as related to the age of the host and the number of oocysts ingested. Poultry. Sci. 34:415-420. Gardiner, J. L. and M. M. Parr. 1954. Nitrofurazone for the prevention of experimentally induced Eimeria tenella infections in chickens. J. Parasit. 40: 42-49. Gardiner, J. L. and D. K. McLoughlin. 1963Drug resistance in Eimeria tenella . III. Stability of resistance to glycarbylamide. J. Parasit. 49:657-659. Glimstedt, G. 1936. Der stoffwechsel bakterienfrier tiere, I. Allgemeine methodik. Skand. Arch. Physiol. 73: 48-62.

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li^3 Waletzky, E. and C. 0. Hughes. 19^9-1950. . Factors involved in tests for acquired immunity in Eimerla tenella infections of the chicken. Ann. N. Y. Acad. Sci. 52:^78-^95. Waletzky, E., C. 0. Hughes, and M. C. Brandt. 19^9-1950. The anticoccidial activity of nitrophenide. Ann. N. Y. Acad. 52:5^3-557Waxier, S. H, 19^1. Changes occurring in the blood and tissues of chickens during coccidiosis and artificial hemorrhage. Am. J. Physiol. 13^:19-26. Weichselbaum, T. E. 19^6. An accurate and rapid m.ethod for the determination of proteins in small amounts of blood serum and plasma. Am. J. Clin. Path. 16:^0-^9. Weinstein, P. P., VJ. L. Newton, T. K. Sav;yer, and I. Sommerville. 1962. Quoted by 17. L. Neviton. In W. I. Gay (ed.). Method of Animal Experimentation, Vol. I, 1965. Academic Press, New York, p. 265. Westcott, R. B. 1971. Experimental infections of gnotobiotlc mice with Nematospiroides dubj.us and Nippo strongylus brasiliensis . J. Parasit. ^6:k'?jZ. Willingale, J. M. and C. A. E. Briggs. 1955. The normal intestinal flora of the pigs. II. Quantitative bacteriological studies. J. Appl. Bact. 18:2b^293. Wittner, M. and R. M. Rosenbaum. 1970. Role of bacteria in modifying virulence of Entamoeba histolytica . Studies of amebae from axenic cultures. Am. J. Trop. Med. Hyg. 19:755-761. Wostmann, B. S. 1959Serum proteins in germ free vertebrates. Ann. N. Y. Acad. Sci. 78:25^1-260. Wostmann, B. S. and E. Bruckner-Kardoss . 1959Development of cecal distention in germ-free baby rats. Am. J. Physiol. 197:13^1-5-13^6.

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BIOGRAPHICAL SKETCH Chittur Venkitasubhan Radhakrishnan was born in Trichur, India, June 6, 1937. He attended primary and secondary schools in Trichur and graduated with first class from Salvation Army English high school, Trivandrum, in March, 1953In June, 1953, he enrolled at the Intermediate College, Trivandrum, and passed the interscience examination with distinction in physics and chemistry in March, 1955From August, 1955, to April, 1959, he attended the Kerala Veterinary College and Research Institute, Trichur", India, and received the degree of Bachelor of Veterinary Science with first class. During his college days he was a member of the soccer, hockey, and field and track teams, and also represented his college in the intercollegiate debate competitions. From July, 1959, to July, 1964, he served as Junior Lecturer at the Kerala Veterinax^ College and from July, 1966, to September, I968, he served as scientific officer at the Antibiotics Research Laboratories, Poena, India. He married the former Jayalakshmy Subramaniara in December, 1965They have one son, Raja Venkitasubhan. Ik-ii

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145 He is a member of the American Society of Parasitologists, the Southeastern Society of Parasitologists, and the honorary fraternities Alpha Zeta and Phi Sigma. He entered the University of Florida in September, 1968, and was granted a research assistantship with the Department of Veterinary Science. At present he is a candidal ^ for the degree of Doctor of Philosophy.

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I certify that I have read this study and that in ray opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for th^^egree of Doctoxy-iDf Philosophy. aici:,:iL'r. i:;. Bradley, Sr., Chapman Associate Professor Department of Veterinary Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. l-g--
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I certify that I have read this study and that in my opinion it conforms to acceptable standards of "scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. '/t^v<-^t^ . Smart, Jr. f Grover G, Associate Professor Department of Entomology and Hematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Sxepiienf/Q. ^am Associate Professor Department of Zoology This dissertation v;as submitted to the Dean of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for th'3 degree of Doctor of Philosophy. December, 1971 Dean, Graduate School


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