Eimeria tenella

MISSING IMAGE

Material Information

Title:
Eimeria tenella comparative pathology and lesions of experimental infections in bacteria-free, specific pathogen-free and conventional chickens
Physical Description:
xi, 143 leaves : ; 21 cm.
Language:
English
Creator:
Radhakrishnan, Chittur Venkitasubhan, 1937-
Publication Date:

Subjects

Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1971.
Bibliography:
Includes bibliographical references (leaves 124-143).
Statement of Responsibility:
by Chittur Venkitasubhan Radhakrishnan.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
oclc - 07267291
ocm07267291
System ID:
AA00011121:00001

Full Text














EIMERIA TENELLA: COMPARATIVE PATHOLOGY AND
LESMIONiS F EXPERIMENTAL INFECTIONS IN
BACTERIA-FREE, SPECIFIC PATHOGEN-
FREE AND CONVENTIONAL CHICKENS





By
Chittur Venkitasubhan Radhakrishnan


A Dissertation Presented to the Graduate Council of
the University of Florida in Partial Fulfillment of
the Requirements for the Degree of Doctor of Philosophy








University of Florida
1971


























To my parents Subhalakshmy and Venkitasubhan

who inspired me to pursue a scientific career.














ACKNOWLEDGMENTS


The author expresses his appreciation and deep sense
of gratitude to Dr. Richard E. Bradley, Sr., chairman of

his Supervisory Committee, for helpful suggestions, counsel,

and encouragement throughout this study and during prepara-

tion 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 student

Mr. David J. Weiner. Mr. Louis N. Ergle and Mr. James E.

Dickinson, Jr., provided technical help for which 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 ways.

The author wishes to gratefully acknowledge the

financial support of the Department of Veterinary Science

in providing a graduate research assistantship which sup-

ported him throughout the study. The research was carried

out as a part of a major project, Hatch 1419 (W-102),

entitled "Biological Methods of Control for Internal


iii









Parasites of Livestock." He is greatly indebted to his

wife, 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.













TABLE OF CONTENTS


ACKNOWLEDGMENTS ..................... .... ........ iii

LIST OF TABLES .................................... vii

LIST OF FIGURES ............................. ix

ABSTRACT ........................................... x

INTRODUCTION ....................................... 1

LITERATURE REVIEW .................................. 8

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 ............................. 28

Pathogenicity of Intestinal Parasites and
Parasitism in Gnotobiotic Hosts .............. 34

NORMAL MICROBIAL FLORA OF CHICKENS ................ 40

MATERIALS AND METHODS ............................. 44

I. Production of Gnotobiotic Chickens ........ 44

II. Production of SPF Chickens ................ 45

III. Production of Conventional Chickens ...... 46

IV. Source of Eimeria tenella ................ 47

V. Pathological Examinations ................. 47

VI. Bacteriological Procedures ................. 49

A. Determination of microbial flora of
the cecum in conventional chickens ..... 49









B. Determination of microbial flora of
the cecum in germ-free, SPF, and
conventional chickens ................. 51

VII. Packed Cell Volume ....................... 51

VIII. Total Serum Protein ...................... 5f

IX. Serum Electrophoresis .................... 53

X. Exposure to Bacteria and Fungi ........... 54

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 Con-
ventional Chickens Inoculated with E. tenella
and Uninooulated Controls ....................... 56

DISCUSSION ......................................... 63

SUMMARY AND CONCLUSIONS ........................... 79

APPENDIX A .................. ............ ....... ... 82

APPENDIX B ........................... .............. 120

BIBLIOGRAPHY ............................... .... 124

BIOGRAPHICAL SKETCH ................................... 144














LIST OF TABLES


Table Page

I Primary Isolation Media Used for the
Recovery and Identification of Bacteria
and Fungi from Ceca of SPF and Con-
ventional Chickens ..................... 52

II Cecal Flora of Young Conventional
Chickens Exposed to Eimeria tenella ..... 83

III Predominant Bacterial and Fungal Species
from Chickens Showing Lesions of Cecal
Coccidiosis and from Nonexposed Con-
ventional Chickens ...................... 84

IV Pathology Due to Eimeria tenella in
Bacteria-, Fungi- and Pleuropneumonia-
Like Organisms-Free Chickens Raised in
Plastic Film Isolators .................. 86

V Pathology Due to Eimeria tenella and a
Single Species of Fungus in Bacteria-
Free 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 .................. 94

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 perfringens
in Three Weeks Old Specific Pathogen-
Free and Conventional Chickens Exposed
to Eimeria tenella ...................... 110


vii











XI Mean Packed Cell Volume (PCV) of
Bacteria-, Fungi-, and PPLO-Free,
Specific Pathogen-Free (SPF) and
Conventional Chickens Exposed to
Eimeria tenella ....................... 112

XII Total Serum Proteins and Serum Protein
Fractions in Infected and Noninfected
Conventional, Specific Pathogen-Free
(SPF) and Bacteria-, Fungi- and PPLO-
Free Chickens ........................ 118


viii


Table


Page














LIST OF FIGURES


Figure Page

1 Ceca 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 sec-
tion of cecum of 3-week-old specific
pathogen-free chicken exposed to
Eimeria tenella showing denudation of
mucosa, and large 2nd generation
schizonts. Hematoxylin-eosin stain.
X125. Microscopic grading of para-
sitism ++++. ....... ........... .............. 121

3 Ceca of 3-week-old bacteria-, fungi-
and PPLO-free chicken exposed to
Eimeria tenella, 7th day post-
inoculation. Macroscopic grading of
the lesion 0. ................................ 122

4 Photomicrograph of a transverse sec-
tion of cecum of 3-week-old bacteria-,
fungi- and PPLO-free chicken exposed
to Eimeria tenella showing a single
coccidium and no denudation of mucosa
or other tissue damage. Hematoxylin-
eosin stain. X400. .......................... 122

5 Ceca of 3-week-old bacteria-, fungi-
and PPLO-free chicken exposed to
Eimeria tenella and Clostridium
perfringens. Macroscopic grading of
lesion ++++. .......................... ........ 123









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 TENELLA: COMPARATIVE PATHOLOGY AND
LESIONS OF EXPERIMENTAL INFECTIONS IN
BACTERIA-FREE, SPECIFIC PATHOGEN-
FREE AND CONVENTIONAL CHICKENS

By

Chittur 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 following experimental inoculation

with a standard dose of E. tenella infective oocysts in

bacteria-, fungi-, pleuropneumonia-like organisms-free

(PPLO-free), specific pathogen-free (SPF) and conventional

chickens were studied.

The dominant microflora of apparently healthy con-

ventional 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 micro-

biological 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 PPLO-

free 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

fecalis or single species of the fungi Candida albicans

or Mucor sp. resulted in mild cecal coccidiosis following

inoculation with E. tenella. The typical cecal coccidio-

sis 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 38% 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. per-

fringens was isolated more frequently from noninfected SPF

chickens than from noninfected conventional chickens. A

stimulation of growth of C. perfringens and coliforms

occurred with a concomitant reduction in the growth of

Lactobacillus sp. in SPP and conventional chickens suffer-

ing 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.













INTRODUCTION


Eimeria tenella [Railliet and Lucet, 1891; Protozoa:
Eimeriidae] is the most common and pathogenic of the 9
species of Eimeria described from the chicken (Gallus
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 known
as coccidiosis.

Coccidiosis is a disease of great economic importance
to the poultry industry throughout the world. In the United
States alone, a total loss to the poultry industry of

$34,854,000 was estimated during the period 1951 to 1960
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
coccidiostatio drugs in the United States was estimated to
be between $40,000,000-$50,000,000 for broiler and laying
flock replacement 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








about 4 weeks of age (Gardiner, 1955). Several other
authors also suggest that young chickens are more sus-
ceptible than older ones (Tyzzer, 1929; Karmann and Presch,

1933). Conversely, many others suggest that the reverse
is true (Tyzzer et al., 1932; Mayhew, 1934; Jones, 1932;
Horton-Smith, 1947). It is important to distinguish between
susceptibility of chickens to clinical disease and sus-
ceptibility to coccidial 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 excysta-
tion of the oocysts (Doran and Farr, 1965; Rose, 196T7).
Older birds are also susceptible to clinical infection but
the rate of mortality is usually low due to an apparent
acquired immunity after previous nonfatal exposures.
Levine (1963) suggested that the acquired immunity is often
not absolute, but generally only a condition of relative
immunity. One-day-old or 1-week-old chickens are less
susceptible to the infection when 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 ex-
posure, 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








to be incorporated in the feed or drinking water 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 with immunity (Davies and Kendall,

1955; Reid, 1960), side effects such as reduced fertility
(Joyner, 1964), and development of drug-resistant strains

(McLoughlin and Gardiner, 1961a, 1961b, 1962; Pellerdy,

1961, 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 contami-
nation in the environment, reduced uptake of the drug or

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

ceptibility (Joyner, 1964, 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 sporu-

late on the ground if conditions of oxygen tension, moisture,
temperature, and other environmental factors are suitable.








The sporulated oocyst, containing 8 sporozoites, 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 4, 5, or 6 weeks

old, while less successful excystation and low oocyst pro-
duction occurred in 0-3-week-old chickens which are also
less susceptible to cecal coccidiosis. She ascribed the
reasons for very low percentage of excystation to im-
maturity of the hosts (weak 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, 1942; Ikeda,

1956; Hibbert et al., 1969).
Our knowledge regarding the role of intestinal flora
in the initiation, development, or severity of cecal cocci-
diosis is limited. However, it has been reported by

Johansson and Sarles (1948) that during E. tenella infec-
tion the growth of Clostridium perfringens is stimulated
while growth of Lactobacillus sp. is suppressed. The
cecal bacterial flora constitutes 90% of the total gastro-
intestinal flora and is of great biological importance to
the health of chickens for synthesis of certain vitamins
(Coates et al., 1968; Timms, 1968).

The effect of normal bacterial flora on the biology
and immunology of the host and possible relationship between





5


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 patho-
genesis of amoebiasis. Based on these findings, Wittner
and Rosenbaum (1970) studied the role of bacteria in modi-
fying the virulence of E. histolytica. Bradley and Reid
(1966) demonstrated a dual etiology involving a protozoan
(Histomonas meleagridis) and a single species of bacteria
(Escherichia coli, C. perfringens, or Bacillus subtlis)
for infectious enterohepatitis in turkeys. Several other
studies have been made on the role of bacterial flora af-
fecting the course of infection in infectious entero-
hepatitis (Doll and Franker, 1963; Franker and Doll, 1964;
Reid et al., 1969; Springer et al., 1970). Hegde et al.

(1969) studied the pathogenicity of E. brunetti in bacteria-
free chickens and showed 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-, fungi- and
pleuropneumonia-like organisms (PPLO)-free) chickens. In
preliminary studies, Clark et al. (1962) found no differ-
ence in the course of E. tenella infection in bacteria-free
and conventional chickens. There was, however, a delay of
12 to 15 hours in the appearance of the 2nd generation









merozoites in the feces of gnotobiotic chicks. Visco and

Burns (1966, quoted by Hegde et al., 1969) reported a close

relationship between 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 whether 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 with 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

SPF 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 pro-

ducing cecal coccidiosis in gnotobiotic chickens were then





7


isolated and the isolates used for further inoculation of
conventional, SPF and bacteria-free chickens. The know-

ledge 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 cocoidiosis.













LITERATURE REVIEW


Eimeria tenella [Railliet and Lucet, 1891] is a
protozoan belonging to the Family Eimeriidae, Class
Sporozoa. The term "coccidia" is generally used to de-

scribe species belonging to the Family Eimeriidae (Becker,

1934). During the last 10 years, studies on the fine
structure of coccidia and related groups have revealed a
great number of new similar structures namely the pellicle,
the polar rings, the subpellicular microtubules, the
rhopteries, the micronemes, the micropore and the conoid.
These fine structural similarities are considered as

indication of a close relationship and Levine (1969) pro-
posed 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 (Biester and Schwarte, 1965). E.
mivati is the only species which may be found in several
regions of the intestinal tract (Edgar and Seibold, 1964).

The 9 species can be differentiated by morphological charac-
teristics, sporulation time of the oocysts, developmental








features, localization in the host, and degree of patho-

genicity. Of the morphological characters, the structure

of the oocyst is usually used to identify the species at

least within a given host (Levine, 1961), 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 develop-
ment of endogenous stages. Also infection with 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 when 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 chickens is regu-
larly associated with E. tenella and this species is the
most common and most pathogenic of all coccidia found in








chickens (Davies et al., 1963). Tyzzer (1929) published

a detailed description of the morphology and life cycle of
E. tenella which has been confirmed in all details by sub-

sequent investigation (Edgar, 1941). Like other species

of Eimeria, asexual and sexual generation occur in the
same host following ingestion of viable sporulated oocysts

through food and/or water. Through a process of excysta-

tion, sporozoites escape from the sporocysts and oocysts,

but the factors contributing to excystation have not been

definitely established. Studies by Levine (1942) and by
Ikeda (1955a, 1955b, 1956, 1960) revealed that pancreatic

juice, in particular trypsin, is one of the factors re-

sponsible for excystation. Goodrich (1944) observed the
escape of sporozoites through any available fracture in

the cyst wall 5-10 minutes after the cyst wall has been

placed in a 5% trypsin solution, maintained at 370 C.

Hydrogen-ion concentration, bile and buffers were also
found to be important factors in excystation of various
species of coccidia (Smetana, 1933; Lotze and Leek, 1960;

Doran and Farr, 1962; Nyberg and Hammond, 1964). 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 excysta-
tion of E. bovis and E. ellipsoidalis in bovine bile con-
taining a heavy suspension of bacteria and fungi. When








trypsin alone was used only E. bovis oocysts excysted, the

other 9 species of Eimeria including E. tenella did not

excyst. The precise way in which bile acts in excystation

of oocysts is not known at present. Doran and Parr (1962)

suggested that bile acids may alter the protein or lipo-

protein 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 (1969) found

that in adult chickens about 40-60 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 wall through which sporozoites might

escape. Therefore the release of large numbers of sporo-

zoites into the digestive tract of chickens requires the

wall of the oocyst to be broken, weakened or partially dis-

solved. Lotze and Leek (1969) observed the walls of many

sporulated oocysts expelled through feces to be structurally

changed. Development of immunity does not hinder excysta-

tion and normal excystation will occur in immune and non-

immune chickens under optimal conditions (Horton-Smith et al.,

1963).

Liberated sporozoites are fusiform, 10 U x 1.5 in
diameter, transparent and motile. Each sporozoite has a

nucleus, a prominent refractile globule at the rounded end,
and exhibits various types of movement. They rapidly invade








the surface epithelial cells of the cecum and then pene-
trate the basement membrane to enter the tunica propria
through which they either pass free or within the macro-
phages, to finally reach the epithelial cells lining the
funds of the Lieberkiihn glands where asexual reproduction
by schizogony occurs (Challey and Burns, 1959; Pattillo,

1959). After entry into a glandular epithelial cell, the
sporozoite rounds up and becomes a trophozoite which
develops into a 1st generation schizont (24 ,u in diameter)
within 24-48 hours. The nucleus of the invaded cell be-
comes hypertrophied (Levine, 1963) and the parasitized
cell bulges out into the cecal lumen. Each schizont forms
about 900 merozoites (Tyzzer, 1929) 2-4 p in length 1-1.5 p
in width. Merozoites after release from the mature 1st
generation schizonts enter a new host cell by direct pene-
tration 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 (1969).
Growth of the 2nd generation schizonts is rapid and within
24 hours mature schizonts, containing numerous merozoites,
can be observed. The large 2nd stage schizonts of E.
tenella are found in the epithelial cells which 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








generation merozoites causes extensive destruction of the

epithelial cells and severe hemorrhage occurs into the cecal

lumen followed by tissue necrosis and thickening of the

cecal wall by the 4th and 6th day. The 2nd generation

merozoites are considerably larger than the 1st, averaging

about 16 in length, 2 ~z in width and 200 to 350 in number,

many of which enter new host cells and begin the sexual

phase of life cycle by developing into either macrogameto-

cytes or microgametocytes. Microgametocytes are smaller

both in size and numbers than macrogametocytes and the 2

are found in close proximity within columnar epithelial

cells of the ceca, below the host cell nuclei. From each

microgametocyte, microgametes develop. Each microgamete

has 3 flagella and is motile.

Young macrogametocytes are large irregularly shaped

cells measuring approximately 5.3 um x 7.5 pm. Though the

young macrogametocyte still retains the shape of the mero-

zoite, it can be distinguished from schizont or microgameto-

cytes by the presence of "wall forming" or membraneous

bodies (Scholtyseck, 1962) under the electron microscope.

Later "dark bodies" develop which are thought to correspond

to the "plastic granules" described from light microscope

investigations (Reich, 1913; Doflein and Reichenow, 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








membrane of the oocyst wall. The middle layer of the dyst
wall 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 wall is trilaminate.
When the oocyst wall is complete the oocyst is extruded
from the host tissues and is passed to the exterior with
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 well 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 H'nig, 1954). The size of the oocysts range
from 14.2 p to 31.2 j x 9.5 p to 24.8 u, with a mean of
22.96 ( 2.2) x 19.16 ( 1.69) p (Becker, 1956). The
optimum temperature for sporulation of the oocysts is
29 1 10 C. (Edgar, 1955) and at this temperature sporula-

tion will be completed in 18 hours but at room temperature
it takes about 48 hours. The sporulated oocysts contain
4 sporocysts each containing 2 sporozoites. Like in other
species of Eimeria 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, 1967, 1969; Strout and Ouellette,








1969; Matsuoka et al., 1969; Doran, 1970), using sporozoites
obtained by in vitro excystation.

Pathogenesis of E. tenella Infection

Factors affecting pathogenicity of E. tenella in-

clude the number of oocysts ingested, the number of host

cells destroyed per ingested oocyst, the degree of re-

infection 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 imper-

ceptible reaction to death (Gardiner, 1955). Cecal cocci-

diosis under field conditions occurs principally 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 weeks (Biester and Schwarte, 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 weeks and 2 months while in older birds

(3, 4, 7, 10, and 15 months of age) mortality was low or
lacking though drop in red cell count ranged from 29% to

46.8%. 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, 4, 5, and 6 weeks.

Those in the 4 week group were severely affected and those

in the 2 week group were least affected. Those birds which









recover from infection become immune to reinfection with

E. tenella. However, 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 (1940) in a study of sub-

clinical coccidial infection in pullets at least 8 months

old reported the presence of E. tenella in 23%. 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 resist-

ance. 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 resist-

ance.

Inherited resistance in some strains of chickens to

infection with E. tenella has been reported by Rosenberg

(1941) and Rosenberg and McGibbon (1948) but in general

there is little evidence of any significant variation in

susceptibility between different breeds or strains of

chickens (Horton-Smith and Long, 1963). Jeffers and

Wagenbach (1969) reported higher susceptibility and mor-

tality of female chick embryos from widely different genetic

sources to E. tenella infection. Edgar and Herrick (1944)

produced evidence to show 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









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 (1929)

postulated that, theoretically, infection with a single

oocyst of E. tenella could give rise to approximately

1,800,000 oocysts in a period of 4 to 5 days. During the

course of an infection, however, there are several factors

which may cause a reduction in this potential including

loss of merozoites, over crowding, and tissue damage which

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 infec-

tion. However, Johnson (1927) reported that the severity

of cecal coccidiosis depends on the number of sporulated

oocysts that the bird receives. Jankiewicz and Scofield

(1934) reported that a dosage of up to 150 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 with individual infections. Fish (1931) reported









that oocysts were not present in the droppings of the in-

fected birds after 17 days although Tyzzer et al. (1932)

recorded oocyst passage for as long as 19 days post-

infection. The greatest numbers of oocysts are discharged

in a very short time (Tyzzer et al., 1932), the few re-

maining 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 (1940) observed oocysts of E. tenella in the drop-

pings 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, the

degree of pathogenicity is related to the depth to which

the cecal wall 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 4th to 7th days

post-exposure. Much of the damage may also come from









secondary bacterial infection of the area in which the

epithelium is completely destroyed (Briggs, 1968). Hem-

orrhage is a great stress on the infected chicken and

feeding and movement are at a minimum during this period,

but consumption of water is increased 2 to 3 times that

seen in uninfected birds. In a typical severe infection,

bloody droppings will 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 90% of the

mortality occurs within 9 days following initial exposure

to oocysts. Chickens surviving 9 days following exposure

will 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 whole, the disease is nearly always of short

duration. It is often found that a condition of disease

arises only when infection, heavy in relation to the pre-

vious 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 world, 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,








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 blood is prevented during the acute stages by
some unknown factors) (Davies et al., 1963) and deficiency

of vitamin K increases pathogenicity to E. tenella and E.

necatrix (Davies et al., 1963). Blood drops can be ex-

pressed 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 diagno-

sis of cecal coccidiosis. The mere presence of oocysts is

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, known
numbers of sporulated oocysts are administered orally.

However, Davies and Joyner (1962) and Sharma and Reid (1962)

succeeded in producing cecal infections by introducing E.

tenella oocysts subcutaneously, intravenously, intra-

peritoneally or intramuscularly in chickens. When viable

sporozoites were introduced by the same routes, light in-
fections 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 (1963) confirmed these findings partially in that









although they could obtain oocysts in the ceca after

intravenous and intramuscular administration of oocysts,

they failed to recover oocysts from ceca when chickens

were inoculated intraperitoneally or subcutaneously. They

assumed that oocysts inoculated into the blood stream would

be removed from the circulation by the liver along with

other foreign bodies. They reported the presence of dis-

integrating 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 were

placed in Millipore chambers grafted within muscles, ex-

cystation 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 sporozoites

to various parts of the body. In all these cases much

lighter infections occurred after parenteral inoculation

compared with the infections occurring after oral inocula-

tion (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 in-

testine will be parasitized. The lesions associated with








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 rela-

tively 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 patho-

logic effects of the maturation of schizonts and release

of 2nd generation merozoites are hemorrhage and sloughing

of the epithelial lining of the cecum, which is sometimes

stripped down to the base of the submucosa and its replace-

ment 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 com-

plete and rapid, but in heavier infections, recovery is

slow and the mucosa often show only incomplete regeneration.

The continuous hemorrhage from the 4th to 7th day post-

exposure results in profound anemia which is often the
cause of death. The exposed skin and mucous membranes









become pallid. Erythrocyte counts and hematocrit decrease

to about 50% of the normal on the 5th and 6th day after

infection with 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 eosinophilia on the 10th day following

infection with 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 (1940, 1941) observed an

increase in blood sugar during the acute stages of the

disease with a decrease in the muscle glycogen. Waxler

(1941) 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 with 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 concentra-

tion on the 1st and 2nd days after exposure and 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 coccidiosis

a severe interference with normal phosphorylative carbo-

hydrate utilization may occur. Challey (1962) noted an








increase in adrenal ascorbic acid and adrenal corti-

costerone concentrations during the acute hemorrhagic

phase of the infection. Bertke (1963) found renal clear-

ance of uric acid in chickens infected with E. tenella

greatest at 2 to 4 days after infection. This study

suggested that death is not due to cecal tissue destruc-

tion 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. Musajev

and Surkova (1970) studied the nitrogen metabolism of

chickens infected with 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 of the parasite. They assume that these disorders

are associated with many factors, such as disorders of

fermentation and suction and influence of metabolic pro-

duct 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 (1954, 1957)
showed that the voluntary muscles in infected birds are
unable to do more than 50% of the work done by muscles of








uninfected birds when stimulated via the nerves. However,
Freeman (1970) found that when the muscles are directly
stimulated they are able to do work and speculated that
an impairment of nervous conduction at the neuro-muscular
junction occurred in coccidiosis.

As for cellular responses, Pierce et al. (1962)
showed that during the primary infection with E. tenella,
heterophilpolymorphonuclear cells infiltrate into the
submucosa in increasing numbers especially on the 4th 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 et al. (1942) first
described a method of scoring E. tenella lesions using a
0 to 4+ scoring system. This scoring system has been
followed by many workers (Ripsom and Herrick, 1945;
Gardiner and Farr, 1954; Cuckler, 1957; Bankowski et al.,

1959; Horton-Smith et al., 1961; Lynch, 1961; Britton
et al., 1964; Turk and Stephens, 1967; Dunkley, 1968). A
modification of this was also used by Cuckler et al.

(1958), Waletzky et al. (1949-1950), Ball (1959), Boney
(1948), Farr and Wehr (1945), Levine and Barber (1947),
Waletzky and Hughes (1949-1950). 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









supplemented by examination for developmental stages in

the cecal contents. Lesion scoring is time consuming.

Should other disease conditions such as ulcerative enteri-

tis appear in the pens, more extensive microscopic studies

may be required to decide whether lesions are induced by

coccidiosis. Norcross and Washko (1970) examined in-

testinal tissues from 734 cases of clinically diagnosed

or suspected cases of coccidiosis histologically and con-

firmed coccidiosis only in 58.2% of the cases. No specific

pathological manifestations could be found in the intesti-

nal 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, helminthia-

sis and histomoniasis. These studies stress the necessity

of both macroscopic and microscopic examination of the

intestinal tissues for confirming pathology due to E.

tenella infection.


Microflora and Hosts

In nature, animals live in intimate contact with

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 symbio-

tic relationships between the host animal and the micro-

organisms. The apparently healthy laboratory and other

animals used to study many biological processes carry








their indigenous microflora and these animals are known as

"conventional" animals. In contrast, a "germ-free" animal

is one from which it is not possible to recover any viable

organism. Many laboratories employ tests to detect bac-

teria, fungi, helminth parasites, PPLO, and certain viruses

to determine the germ-free state of the animals (Newton,

1965). The term "gnotobiote" (Reyniers et al., 1949) 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 micro-

organisms and parasites known to cause disease (Sabourdy,

1965). The SPF animals are functionally and structurally

identical with their conventional counterparts but their

flora and fauna are, to some extent, controlled. The

production of germ-free or SPF animals is a problem if the

particular animal species have certain congenital infec-

tions. Salmonella pullorum in chicks and Toxocara canis

in dogs (Reece et al., 1968; Griesemeret al., 1963) are

two infections transmissible congenitally. Careful selec-

tion and isolation of breeding stock free of these infec-

tions and prompt elimination of young animals or chicks

showing any congenital infections are necessary to insure

absence of such infections in the breeding stock (Reece

et al., 1968). These elaborate procedures to establish

and maintain germ-free or specific pathogen-free animals

for research purposes will eliminate the necessity for

using experimental animals of unknown disease exposures,









age, breeding and most important environmental background.

The possible influence of the "macro" and "micro" environ-

ment in disease processes can be studied when 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 germ-
free animals since Pasteur's speculation in 1885 that the

host-microflora relationship is obligate. But the in-

vestigations of Schottelius (1899), Cohendy (1912),

Cohendy and Wollman (1914), Kister (1912), Glimstedt

(1936), Balzam (1937a, 1937b), Reyniers (1946, 1949t
1960), Gustafsson (1948) and Miyakawa (1954) proved

Pasteur's original assumption wrong. Now the germ-free

animal has become a very useful tool for studying true

homeostasis of the gnotobiotic host, the individual action

and interactions of microorganisms and the response of the

host to these organisms. These interactions have not yet

been well defined although a few significant microflora-

host 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 with relative ease. Germ-free chickens are
very popular as experimental animals and have been









successfully used to study such research problems as 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 et al., 1949, 1960).

The germ-free chickens are less clean than con-

ventional chicks due to the high humidity in the units

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 gastrointestinal tract

are altered in the absence of viable flora in the germ-

free animals (Reyniers, 1960). The intestinal mucosa

had less lymphatic development and less connective tissue

mass in germ-free chickens and the general picture was

that there were more absorptive elements but fewer and

less well developed elements of defense (Reyniers et al.,

1960). Gordon (1960) found about 3 times greater numbers

of reticulo-endothelial cells in the mucosa and the sub-

mucosa 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 lympho-

cytes in the submucosa and lamina propria of the lower









ileum. However, the epithelial cell content was greater

in germ-free chicks. The amount of lamina propria in the

total area studied was 25.6 0.9% in germ-free chicks

and 36.8 1.4% in conventional chicks. Eyssen and

DeSomer (1967) reported that the weight of the small

intestine was 105% greater in conventional than in germ-

free chicks. Enlargement of the cecum was 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 was

not observed in germ-free chickens and turkeys (Luckey,

1963). The ceca of the germ-free chickens were found to
be significantly shorter in length than those of chickens

with a normal bacterial flora (Hegde et al., 1969). From

gross observation, the large intestine and cloaca of con-

ventional and germ-free chicks were similar. However,

the relative wet weight of large intestine per 100 grams

body weight was greater in germ-free Leghorn chickens.

The feces of germ-free and conventional chicks were grossly

similar, but the germ-free chicks were 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 et al., 1960; Gordon,

1960) between germ-free and conventional chickens. The

ileo-cecal tonsils of conventional chickens were of larger









size, full and more turgid while ileo-cecal tonsils of

germ-free birds were flabby, pale and inconspicuous. This

difference persisted till 5 months of age and from then on

the difference became less apparent. The relative weight

of the trident at the ileo-cecal valve which contains both

the ileo-cecal tonsils was 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 was from 1/5 to 1/20 of that found

in birds harboring live bacteria. Thorbecke (1959) found

no plasma cells or secondary nodules in the ileo-cecal-

colic junction of germ-free chicks at 2, 4, 8, and 14

weeks. They were found in conventional chicks of all ages

and in germ-free chicks at 6 weeks of age. In White

Wyandotte Bantam chicks, the bursa of Fabricus of con-

ventional chicks were larger per 100 grams body weight than

that of germ-free chicks. In germ-free chickens the spleen

was of smaller size (Reyniers et al., 1960) but the general

structure, color and consistency of the peripheral and cut

surfaces were identical with that seen in conventional

groups. Distribution of plasma cells was 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

was found to be comparable with control chicks reared on









natural commercial diet, but the growth rate is reported

to be faster in germ-free chicks (Forbes and Park, 1959;

Forbes et al., 1959). Reyniers et al. (1960) 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 were identical in germ-free and

conventional chickens, but leukocytes were 2 to 5 times

more numerous in conventional chickens. Circulating lympho-

cytes were 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 germ-free animal (Balish and
Phillips, 1966; Thorbecke et al., 1957). The gamma-1,

gamma-2 and beta fractions of the globulin fraction of the

blood of germ-free chicks were 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 known if the low gamma globulin of

the germ-free chicks is "innate" or produced as a result

of an unknown antigenic stimulus. Boggs et al. (1967)

studying granulocytopoiesis in germ-free mice reported a

lower concentration of neutrophils in blood of germ-free

mice but the presence or absence of microorganism did not
alter the overall granulocytopoiesis.

A more positive oxidation-reduction potential of








cecal contents appears directly related to the absence of

gastrointestinal microflora. Balish and Phillips (1966)

and Springer (1968) found the oxidation-reduction poten-

tials of bacteria-free cecal contents of the chicken

strongly positive while those of the conventional chicken

were strongly negative. Balish and Phillips (1966) re-

ported that the pH was higher in all segments of the gut

in germ-free chicks when compared to that seen in conven-

tional chicks. The above studies indicated that the germ-

free chicks show acceptable normal growth and reproduction.

In addition, perosis 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" was re-

ported by Gordon et al. (1959) in germ-free chicks due to

cellular proliferation in the brain.

The serum of germ-free animals has been shown to have

a low globulin content and very few anti-bacterial agglu-

tinins, though complement and heterohemagglutinins 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 clear-

ing injected particles or dead bacteria (Luckey, 1963).

The pathology of many infectious diseases has been studied
in germ-free animals.








Pathogenicity of Intestinal Parasites and Parasitism
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 towards 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 when

species of bacteria such as Escherichia coli and Aerobacter

aerogenes are present. However, another parasitic proto-

zoan, Pentatrichomonas (Trichomonas) hominis, developed in

large numbers in germ-free guinea pigs (Phillips, 1962).

Pentatrichomonas (Trichomonas) vaginalis, when subcutan-

eously 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 (Newton et al., 1960).

Experimental infection of gnotobiotic mice with
Nematospiroides dubius and Nippostrongylus brasiliensis









have been produced and studied (Newton 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 following nema-

tode infection, but no eosinophilia was seen in conventional

mice. Nodule development in the intestinal wall 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. Weinstein et al. (1962) reported

that the larvae of N. dubius will not develop to the in-

fective stage in feces from germ-free mice as they normally

do in feces of conventional mice. At least in this case,

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. (1967) and Rohovsky and Griesemer

(1967) found feline infectious enteritis in the germ-free

cat is a mild, nonfatal disease with symptoms of leuko-

penia, thymic atrophy and lymphoid depletion, but without

morphologic intestinal lesions and clinical signs. In SPF

cats, clinical signs, ultrastructural alteration of the

intestinal mucosa and reduced enzyme activity were noted

(Johnson et al., 1967; Fowler and Rohovsky, 1970).









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. gallinarum or the protozoan H. meleagridis. Subse-

quently Bradley et al. (1964) and Bradley and Reid (1966)

demonstrated a dual etiology involving a protozoan (H.

meleagridis) and a single species of bacteria (E. coli

C. perfringens or B. subtlis) for infectious enterohepa-

titis in turkeys. Springer et al. (1970) reported that

the bacterial requirements for producing infectious entero-

hepatitis 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 environ-

ment suitable for the survival of H. gallinarum since

histomonads have been found to survive in bacteria-free

hosts. An enhancement of virulence cannot be overruled in

this infection as in the case of human amebiasis. In his

early studies on the ameba-bacteria relationship Phillips

(1964) stated that it was 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 (1966) have demonstrated

that various bacterial species alter the virulence of E.

histolytica grown in ameba-trypanosome cultures, as








measured by subsequent inoculation into animals. Wittner

and Rosenbaum (1970) studying the role of bacteria in

modifying the virulence of E. histolytica found that the

increased virulence is associated only with contact of

ameba with live bacteria and speculated transfer of an

episome-like virulence factor from bacteria to the proto-
zoans.

Reid and Botero (1967) reported the growth of the
cestode, Raillietina cesticillus, in bacteria-free chickens

and concluded that no contribution to the establishment of

the tapeworn or interference from the normal bacterial

flora of the digestive tract occurs. Johnson (1971) re-
ported the growth and development of Ascaridia galli in

gnotobiotio chickens and the data indicate an inhibition

of development of the nematode in the bacteria-free

chickens.

On the other hand, Balish and Phillips (1966) re-
ported that oral challenge with Candida albicans resulted

in crop infection in all bacteria-free chicks but no in-
fection occurred in conventional chicks. Layton and
Simkins (1971), in their studies with Mycoplasma galli-

septioum, found higher mortality (63%) in germ-free chicks
than in conventional chicks (38%). In gnotobiotic swine,

Kohler and Cross (1969, 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








due to E. coli in germ-free pigs. These studies all

indicate that normal flora has either a beneficial or

antagonistic action on many of these pathogenic organ-

isms. Another intriguing role for the associated micro-

flora in the host-parasite relationship, perhaps in

determining host specificity, was the resistance of the

conventional guinea pigs to Trypanosoma cruzi. However,

a majority of the germ-free guinea pigs harbored the

trypanasomes in their blood following intracecal inocula-

tion (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., 1969). Clark et al. (1962) also found very

little difference in the pathogenicity of E. tenella in

conventional and bacteria-free chickens although there

was 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 (1966,

quoted by Hegde et al., 1969) reported no mortality in 41

gnotobiotic chickens infected with E. tenella as compared

to 77% mortality in infected conventional chickens. They
concluded that a close relationship exists between the

host flora and the protozoan in the production of cecal

coccidiosis syndrome. Kemp et al. (1971) reported a

delayed development of endogenous stages of E. tenella

in germ-free chicks, especially 2nd generation schizonts,

gametocytes and oocysts. There was a striking lack of





39


reticulo-endothelial cells in the lamina propria and

submucosa and substantially low numbers of mononuclear

inflammatory cells. Thus, the effects of the bacterial

flora on the pathogenicity of this species remain

unresolved.













NORMAL MICROBIAL FLORA OF 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 well

documented in the literature (Smith and Crabb, 1961;
Willingale and Briggs, 1955; Dubos and Schaedler, 1962;
Smith and Jones, 1963; Savage and Dubos, 1968; Ogata and

Morishita, 1969; Savage et al., 1968, 1970; Rall et al.,

1970). In avian species, the microflora studies have
been limited to turkeys and chickens.

Cook et al. (1954) studied the effects of anti-
biotics on the intestinal microflora of turkey poults.
Naqi et al. (1970a, 1970b) studied the intestinal micro-

flora of normal healthy turkeys from 1 day to 8 weeks of
age and also in those infected with "bluecomb." 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 within the first 24 to 48
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









normal bacterial flora of conventional chickens (Johansson

et al., 1948; Shapiro and Sarles, 1949; Lev and Briggs,

1956; Lev et al., 1957; Huhtanen and Pensack, 1965; Smith,
1965a;Timms, 1968; Barnes and Impey, 1968). 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., 1948; Smith, 1961; Smith, 1965a, 1965b;
Smith and Crabb, 1961). All these studies indicated that
the numbers of bacteria of all groups were found to be
highest in the cecal contents and progressively lower

numbers in the posterior large and anterior small intes-
tine, respectively. The organisms constituting the major

part of the flora were E. coli, enterococci (Streptococcus
fecalis), Lactobacillus sp., Bacteroides sp. and C. per-
fringens. 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 were of particular interest to the
investigators. Shapiro and Sarles (1949) 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








count of enterococci and aerobic bacteria. These were
replaced by anaerobic types at around 14 days of age.

The normal bacterial flora has been reported to
influence the host-specificity of some parasites (Newton

et 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 (1966) reported that in C. albicans
infection of the crop, the count of enterococci was in-
creased. Naqi et al. (1970a)reported significant differ-
ences in the intestinal microflora in turkeys inoculated

with an infectious enteritis ("bluecomb") agent and un-
infected control turkeys. The changes were characterized
by a rise in total microbial count of the entire intestine,

a significant increase in number of coliforms, 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 (1963,

1967) and Ogata and Morishita (1969) in pigs inoculated
experimentally with an enteric pathogen. Johansson and
Sarles (1948) noted that in E. tenella infection, a stimu-
lation of the growth of C. perfringens occurred with con-
current 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 interference in glucose metabolism









and a role for the flora in the pathogenesis of cecal

coccidiosis. Thus, in cecal coccidiosis the problem to

be studied is whether 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 epithe-

lium, development of schizonts and/or gametocytes and

thereby contribute to the tissue damage.














MATERIALS AND METHODS


I. Production of Gnotobiotic Chickens

Gnotobiotic (bacteria-, fungi- and PPLO-free)

chickens were raised in flexible plastic flim isolators

following the method of Bradley et al. (1967). All

isolators, accessories and supplies were obtained from

the same source.1 The methods employed in sterilization,

maintenance, and operation of gnotobiotic environment

chambers and equipment, the sterilization of feed sup-

plies and the scheme used for determining the micro-

biological status were the same as those described by
Bradley et al. (1967).

Day-old or 19-day-old embryonated White Leghorn
chicken eggs were obtained from a commercial hatchery2

free of Salmonella and Mycoplasma infection and incubated
at the laboratory. Prior to introduction into the isolator

chambers, all eggs were candled at least twice 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

1G. F. Supply Division, 431 North Quentin Road,
Palatine, Illinois 60067.
2Florida State Hatcheries, Gainesville, Florida
32601.









mercuric chloride held at 370 C. The eggs were 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 was placed in a room held at

370 C and 80-85% relative humidity for hatching. After

hatching, the egg shells were removed and the chickens

transferred to a plastic wire-floored basket inside the

isolator. Sterile feed and water were provided ad libitum.

II. Production of SPF Chickens

Chickens hatching from fertile eggs obtained from

the same source as that from which eggs were obtained for

production of gnotobiotic chickens were immediately trans-

ferred to modified Horsfall-Bauer units. Altogether 10

such units were kept in a room adjoining those in which

the plastic film isolators were kept for the production

of gnotobiotic chickens. Air entering the Horsfall-Bauer

units was sterilized by passage through sterilized fiber-

glass filter media. Sterile water was supplied in gallon-

sized 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 was of composition meeting National Research

Council (NRC) standards. The feed was pasteurized in a

hot air oven at 1500 C for at least 60 minutes and was
supplied to the chickens in a metal self-feeder inside









each unit. Temperature was controlled electrically and

ventilation was fan-forced, negative pressure. Before

and after each use, the units were cleaned and scrubbed

with hot detergent solution and steam sterilized. As far

as possible, the units were opened only 3 times during an

experiment--to enter newly-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 follow-

ing specific pathogens:

1. the 9 species of Eimeria causing coccidiosis

in chickens;

2. the common intestinal helminths of chickens

(Ascaridia, Heterakis, and Raillietina

species);

3. Salmonella and Pasteurella species; and
4. H. meleagridis.

III. Production of Conventional Chickens

Newly-hatched chickens were transferred from the
incubator and reared in electrically-heated battery brood-

ers. Unsterilized chicken starter mash, with no anti-

biotics or added chemicals, satisfying NRC requirements,

and water were made available ad libitum. Before and

after each use the pens were cleaned, scrubbed with hot

detergent solution, and steam sterilized.








IV. Source of Eimeria tenella

The same strain of E. tenella isolated from a
natural case of cecal coccidiosis was used throughout
this series of experiments. Oocysts were produced

according to need in disease-free 3-week-old conventional
chickens. Fecal material from donor chickens was col-
lected at 7-9 days after inoculation with a sublethal
dose of oocysts and the fecal debris and other gross
particles removed by sieving through 30 and 80 mesh
sieves. The oocysts were then sedimented by centrifuga-

tion and allowed to sporulate in 2% potassium dichromate

solution at room temperature and after sporulation were
stored at 5 C. Immediately before use in experimental

trials, all oocysts were surface-sterilized with a 0.5%
solution of peracetic acid (Doll et al., 1963). Sterility
was 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. Pathological Examinations

Chickens inoculated with oocysts were kept under
close observation. Bleeding or any other clinical signs
were noted. Blood samples for packed cell volume deter-
mination and serum analysis were taken both before








inoculation and on the 7th day after exposure. Bacterial

isolates were made on both the control and experimental

chickens at necropsy, according to the bacteriological

procedure described below. Chickens dying after exposure

to oocysts and all those surviving on the 7th day after

exposure were 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 examina-

tion, cecal tissues showing gross lesions and cecal

tissues from comparable sites from chickens showing no

gross lesions were fixed in neutral 10% formalin and

sectioned at 5 or 10 1 thickness and stained with

hematoxylin-eosin. The system of macroscopic grading

of lesions and microscopic grading of parasitism was

modeled after Horton-Smith et al. (1961) as follows:

Scheme of Grading of Lesions and Parasitism

Extent of Macroscopic Microscopic
Lesions and Grading of Grading of
Parasitism Lesions Parasitism

0 No detectable No coccidial stages
lesions found even on care-
ful search
+ Small number Small number of
of lesions gametocytes found
by careful search
(5 or less)









Extent of
Lesions and
Parasitism

++


Macroscopic
Grading of
Lesions

Moderate number of
lesions with some
hemorrhage


Numerous lesions
and hemorrhage



Numerous lesions
with severe hemor-
rhage 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 widely dis-
tributed gametocytes
in localized foci with
appreciable tissue
damage

Numerous schizonts
and/or gametocytes
with widespread tissue
damage


VI. Bacteriological Procedures

A. Determination of microbial flora of the cecum in
conventional chickens

The development of cecal microbial flora in disease-

free young conventional chicks and those inoculated with

100,000 sporulated oocysts was studied as follows. Newly-

hatched White Leghorn chicks were raised on standard

electrically-heated battery brooders and fed unsterilized

feed and water ad libitum. Four uninfected control and 4

infected chickens were necropsied at various age intervals:

day-old (control only), 6-day-old, 1, 2, 3, 4 and 5 weeks

of age. Inoculation with oocysts was adjusted so that the

day of necropsy would be 7 days after exposure. At

necropsy, the viscera was exposed and the cecal pouches

together with 2 inches of anterior small intestine and








posterior large intestine were transferred to a sterile

petri dish. Using aseptic procedures, the cecal pouches

were opened, the sterile tip (0.0025 cm2) of an inoculat-

ing 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 media.

In case of dry cecal contents or a formed cecal core, a

drop of sterile pH 7.0, 0.067 M phosphate buffer was used

to soften the material prior to insertion of the sampling

loop. Aerobes were enumerated on DifcoR1 brain heart

infusion agar; enterococci in DifcoR azide-dextrose broth

supplemented with 10 p.p.m. methylene blue and 1.5% agar;

and coliforms on MacConkey agar. The medium used for

determining the anaerobic populations was DifcoR brain

heart infusion agar supplemented with 0.1% cysteine-

hydrochloride and 0.1% bovine serum. C. perfringens was

enumerated on DifcoR sulphite polymyxin sulphadiazine agar.

For fungus isolation, DifcoR Sabouraud dextrose agar and

DifcoR Pagano-Levine agar with antibiotics were used; for

protozoan isolation,DifcoR Balamuth medium was used. Time

of incubation was 18 hours for coliforms, 24 hours for

clostridia and 48 hours for all other organisms. After

incubation, the colonies from each of the 4 uninfected

controls and the 4 infected ceca were counted and mean
numbers calculated.

1Difco Laboratories, Detroit, Michigan 48232.








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-free, SPF and conventional

chickens are listed in Table I. The criteria listed by

Breed et al. (1957) were employed for characterization,

identification, and verification of bacterial species.

Anaerobic plates were incubated in a GaspackRB Anaerobic

Jar #60410 using GaspackR gas generator envelopes for

generating hydrogen and carbon dioxide gas. Methylene

blue was used as the indicator.

VII. Packed Cell Volume

Hematocrit values were determined by drawing the

blood into a heparinized microcapillary tube and centri-

fuging the blood in an International Microcapillary

Centrifuge2 for 5 minutes at 15,000 r.p.m. The packed

cell volume (PCV) was directly read using an International

Capillary Reader.2

VIII. Total Serum Protein

Total serum protein was determined by the method of
Weichselbaum (1946) with slight modifications. A standard


1Baltimore Biological Laboratory, Baltimore,
Maryland.

2International Equipment Company, Needham Heights,
Massachusetts.










Table I
Primary Isolation Media Used for the Recovery
and Identification of Bacteria and Fungi from
Ceca of SPF and Conventional Chickens



DifcoR azide-dextrose broth with 10 p.p.m. methylene blue
and 1.5% agar
DifcoR blood agar with 0.5% defibrinated bovine blood

Difcd brain heart infusion agar with 0.1% cysteine-Hcl
and 0.1% bovine serum
DifcoR Brewer's anaerobic agar

DifcoR blood agar with 0.5% blood agar and neomycin
DifcoR-Columbia broth

DifcoR enterococci presumptive broth
DifcoR MacConkey agar

DifcoR Pagano-Levine base with triphenyl tetrazolium
chloride and antibiotics
DifcoR PPLO broth with antibiotics
DifcoR Sabouraud dextrose agar with antibiotics
DifcoR sulphite, polymyxin sulfadiazine agar

DifcoR Salmonella-Shigella agar

DifcoR thioglycollate medium without dextrose
DifcoR tomato juice agar special
DifcoR trypticase soy broth








curve was plotted using various dilutions of a 10% bovine

serum albumin solution and corresponding optical density

in a Turner Spectrophotometer at a wave length of 540 nm.
In the procedure, 0.1 ml of unknown serum and 8.0 ml of

stable biuret reagent2 were mixed and incubated at 370 C

for 45 minutes. The optical density was then read at

540 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 was done by
MicrozoneR Electrophoresis3 on cellulose-acetate membranes
using pH b.6, 0.075 ionic strength barbital buffer. Using

the applicator, 0.50 yl 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 8 minutes and cleared in 33%

cyclohexanone in absolute alcohol for 1 minute. After

drying, the membrane was scanned in a MicrozoneR Densito-

meter4 and the chart tracings evaluated to obtain the

component percentages.


1G. K. Turner Associates, Palo Alto, California.

2Hycel, Inc., Houston, Texas.
3Beckman Instruments, Inc., Fullerton, California.

Ibid.









X. Exposure to Bacteria and Fungi

Single species or combinations of bacteria or fungi
to be used along with E. tenella for experimental exposure

were selected on the basis of microbial isolates from ceca

of conventional chickens having very severe coccidiosis.

Approximately 0.5 ml of 24 to 48 hour broth culture of

the respective organism was 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 grown in DifcoR Sabouraud dextrose broth at 370 C for

48 hours and 0.5 ml of the broth was administered orally.
Bacterial species were administered 24 hours before E.

tenella oocysts were given; fungal suspensions were given
48 hours prior to oocyst administration.














RESULTS


Development of Microbial Flora in the Cecum of Conventional
Chickens Inoculated with 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 were predominantly enterococci (S. fecalis) with

E. coli making up most of the remainder. In noninfected

chicks at 7 days of age, enterococci were not found to be

the predominant organisms in the ceca, these being replaced

by E. coli and Lactobacillus sp. In E. tenella-infected

chickens, the enterococci were greatly outnumbered by

other species, especially E. coli and Bacteroides sp., but

numbers of Lactobacillus sp. were greatly reduced as com-

pared to noninfected controls.

On the 14th day of age in noninfected conventional

chicks, E. coli and Lactobacillus sp. were predominant

with few enterococci,and Bacteroides sp. were rare. In

the infected chicks, E. coli was the most predominant

species, and Lactobacillus sp. were not detected.









At 21 and 28 days of age, E. coli and Lactobacillus

sp. predominated in the noninfected group while in the

infected group, E. coli, Bacteroides sp., enterococci and

Lactobacillus sp. were detected. A stimulation of growth

of E. coli, Bacteroides sp. and S. fecalis was observed

during E. tenella infection while the growth of Lacto-

bacillus sp. was suppressed. At 35 days of age, the same

pattern was observed except that in chickens infected with

E. tenella, C. perfringens and anaerobic fecal streptococci

were also detected.

Fungal isolates were rare. In 6-day-old non-

infected chickens, Candida and Mucor species were 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 groups and in 2-week-old infected chickens

Mucor species were detected. In 4-week-old chickens, no

fungi were detected in either noninfected and infected

groups.

Bacterial Isolates in 3-Week-Old SPF and Conventional
Chickens Inoculated with 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 shown in Tables VIII and IX. C. per-

fringens was isolated from infected and noninfected SPF

chickens and from infected conventional chickens more fre-

quently than from noninfected conventional chickens. A









stimulation of growth of C. perfringens was noted both

in infected SPF and conventional chickens (Table X). In

noninfected conventional chickens, C. perfringens was

isolated in only 2 trials while in infected chickens this

species was isolated in all the trials. The number of

colonies of C. perfringens developing from a standard

inoculum was higher for SPF chickens than for conventional

chickens. Occurrence of fungal species was also frequent

in SPP chickens, especially in the noninfected ones.

Candida species were isolated from noninfected controls in

4 of the 8 trials, while Candida species were isolated

from only I of the 8 trials in conventional chickens

(Tables VIII and IX). Mucor species were infrequent and

were isolated only in 1 trial from a noninfected conven-

tional chicken inoculated with E. coli, C. perfringens

and S. fecalis.

Pathology due to E. tenella in bacteria-, fungi-

and PPLO-free chickens is reported in Table IV. Clinical

symptoms like bleeding, anorexia, weakness, and drooping

were not noted in any of these chickens. No mortality

occurred in 32 of these chickens raised bacteria-, fungi-

and 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 Fabricus, spleen, heart, muscles,









and lungs were normal as compared to the viscera from

noninfected controls raised bacteria-, fungi-, PPLO-free

and also those raised specific pathogen-free and con-

ventionally. Histopathologically, there was 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 endoge-

nous stages were identical to immature schizonts and

early gametocytes. No large 2nd generation schizonts

containing mature merozoites were seen. Cecal coccidiosis

as described by Tyzzer (1929) and Tyzzer et al. (1932) was

not seen in these chickens when exposed experimentally to

a standard inoculum of E. tenella oocysts.

There was no decrease in hematocrit values in

bacteria-, fungi- and 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.









In chickens harboring Mucor species, moderate

numbers of lesions with some hemorrhage and thickening

was noted macroscopically. Histologically, there was

moderate tissue damage and large numbers of endogenous

stages including oocysts were seen.

In chickens monocontaminated with anaerobic fecal

streptococci and exposed to E. tenella there were no

visible lesions or hemorrhage, but histologically, endoge-

nous stages were evident. Tissue damage, sloughing of

mucosa, hemorrhage, and large 2nd generation schizonts

were also absent. Hematocrit values increased from a pre-

exposure volume of 17.0 to a post-exposure volume of 21.5.

In chickens monocontaminated with anaerobic fecal strepto-

cocci and a suspension of killed E. coli_ Q. perfringens, and

S. fecalis, infection with 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 were seen in the

epithelial cells of the mucosa. The hematocrit values were

27 at day of inoculation and 31 on the 7th day post-infection.

In chickens monocontaminated with either Bacteroides

sp., C. perfringens, E. coli, or other coliforms like A.

aerogenes, moderate numbers of lesions with hemorrhage were

noticed (Table VI). Death due to cecal coccidiosis occurred

only in 2 out of 12 chickens monocontaminated with S.

fecalis, and no mortality occurred in those monocontaminated









with other species of bacteria. In chickens monocontami-

nated with S. fecalis there was a tendency for the blood

to coagulate more rapidly. Partially coagulated blood in

the ceca was characteristically present in these chickens.

Hematocrit values also showed a reduction in chickens

monocontaminated with bacteria and infected with E. tenella

(Table XI). Comparatively, chickens monocontaminated

either with Bacteroides sp., C. perfringens or S. fecalis

developed moderate to severe pathology when exposed to E.

tenella. Chickens monocontaminated with E. coli or Lacto-

bacillus sp. developed only mild to moderate lesions, and

those monocontaminated with anaerobic fecal streptococci

showed little pathology when exposed to E. tenella.

Chickens infected with E. tenella and polycontami-

nated with 2 or more species of bacteria or fungi showed

lesions intermediate between those of monocontaminated and

SPF or conventional chickens (Table VII). Heavy mortality

(80%) 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

.. 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









of bacteria and fungi favored development of moderate

lesions, but did not favor development of greater pathol-

ogy. However, the pathology was more severe than that

seen either with the bacterial species alone or fungus

species alone.

Pathological manifestations in E. tenella-infected

SPF chickens were typical of cecal coccidiosis (Table VIII).

Clinical symptoms like bleeding, anorexia, droopiness and

mortality were noted. Thirty-three of the total 88

chickens inoculated died, establishing a mortality rate

of 38%. Macroscopic as well as microscopic lesions were

observed in all infected chickens establishing an infec-

tion rate of 100%. The mean gross lesion score was 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 SPF chickens (Table IX).

Infection rate was 100% and mean gross lesion score was 3.1.

Twenty-three out of 104 inoculated chickens died due to

cecal coccidiosis registering a mortality rate of 22.1%.

The mean hematocrit value dropped from a pre-exposure

volume of 30.3 to 20.1 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 (1929) and Tyzzer









et al. (1932). In cases having a gross lesion score of ++++

or+++, endogenous stages were seen extending deep in the

muscle layers of the cecal wall 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-,

fungi- and PPLO-free chickens are reported in Table XII.

In conventional and SPF chickens infected with E. tenella

there was a consistent reduction in total serum protein

concentration while such reduction was not seen in bacteria-,

fungi- and PPLO-free chickens exposed to E. tenella, when

compared to the total serum protein concentration of non-

infected controls.













DISCUSSION


The cecal microflora was 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 micro-

flora 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 (1948) 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 week-old or older chickens. These results, as well

as the occurrence of large numbers of Lactobacillus sp. and

E. coli in chickens aged 14 to 35 days old, and the con-

stant isolation of Bacteroides sp. agree with the results

obtained by earlier workers in chickens.(Huhtanen and

Pensack, 1965; Timms, 1968), and in turkeys (Naqi et al.

1970b).
Rall et al. (1970) studied the distribution of bac-
teria in feces of swine and identified E. coli, other

lactose fermenters, lactose nonfermenters, Staphylococcus









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 (1964) reported that in soil, bacteria

occur as colonies and Savage et al. (1968) also reported

the occurrence of colonies of bacteria on the walls of the

gastrointestinal tract in rodents. Kolacz et al. (1970)

using conventional dilution techniques noted more varia-

tion 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. Graber

et al. (1966) reported successful isolation of Bacteroides

sp. from SPF swine after vigorous swabbing 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 cm2) was used in

this study. This technique has been successfully employed

by Rall et al. (1970).

The very rare occurrence of Clostridium sp. in

apparently healthy conventional chickens in this study

agrees with the findings of Huhtanen and Pensack (1965).

This rare occurrence of species like C. perfringens which,








though ubiquitous in nature, has been variously explained.

Lev et al. (1957) reported that C. welchii (C. perfringens)

was present in day-old chicks in "infected* quarters but
not in "uninfected" quarters. They established a correla-
tion between 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. welchii (q. perfringens) from ceca of chickens 18 days,

7 weeks, and 5 months old. These species were, however,
absent in the small intestine.

In the present study, C. perfringens was isolated
from SPF chickens frequently. These SPF chickens were

healthy and no "infection" was noticed. They were raised
on pasteurized feed and water. Also, Bacteroides sp. were
isolated from these SPF chickens as well as from conven-

tional chickens. Moreover, in agreement with the report
by Johansson and Sarles (1948), in chickens exposed to E.
tenella, a stimulation of growth of C. perfringens occurred








both in SPF and conventional chickens after exposure to E.

tenella. In conventional chickens with cecal coccidiosis,

C. perfringens was regularly isolated, in contrast to non-
exposed 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 inocu-

lated with 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 between bacteria, competition for common

nutrients, and changes in pH and oxidation-reduction

potential may play an important part (Hentges, 1967, 1969;

Lev et al., 1957; Meynell, 1963; Schaedler et al., 1965).

The present study confirms the observation of Johansson

and Sarles (1948) that growth of C. perfringens 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-, 14-day-, 21-day-, 28-day- and 35-

day-old chickens with cecal coccidiosis, a suppression

of growth of Lactobacillus sp. and a stimulation of growth

of Bacteroides sp., 0. perfringens, and E. coli was noted.

The factors) responsible for suppression of an anaerobic

species during cecal coccidiosis which 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, factors) favoring









growth and multiplication of C. perfringens may be readily

available with a concomitant reduction of those favoring

growth and multiplication of Lactobacillus sp. A differ-

ence in severity of the cecal coccidiosis syndrome was

noted between chickens monocontaminated with C. perfringens

and chickens monocontaminated with Lactobacillus sp. In

the former, the pathological manifestations were more

severe than the latter. Also, more severe pathology

developed in chickens monocontaminated either with S.

fecalis, E. coli or Bacteroides sp. than in those mono-

contaminated with Lactobacillus sp. when exposed to standard

doses of E. tenella oocysts. This indicates that the in-

fluence of microbial flora in the pathology of cecal

coccidiosis depends upon the species involved. This is

much more clearly evident when the pathological manifesta-

tion in chickens monocontaminated with anaerobic fecal

streptococci and also those monocontaminated with C.

albicans is compared with that seen in chickens mono-

contaminated with other species of microorganisms. Little

pathology was noted in chickens harboring only anaerobic

fecal streptococci and inoculation of heat-killed E. coli

C. perfringens and S. fecalis did not influence the de-
velopment of symptoms or pathology.

Pathology and clinical symptoms were negligible in

chickens monocontaminated with C. albicans and only mild

to moderate lesions developed in chickens monocontaminated

with Mucor species. Under natural conditions, fungi seem









to have little influence in the development of cecal cocci-

diosis. In healthy conventional chickens, fungi occurred

only infrequently. This may be due to the antagonism

between the fungi and bacteria which is well known 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 SPF chickens, C. 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 SPF and conventional environments.

A comparison of the pathology of cecal coccidiosis

in SPF, conventional, and polycontaminated chickens raised

in plastic film isolators clearly shows the influence of

the environment and microflora in cecal coccidiosis syn-

drome. The mortality rate from coccidiosis was higher in

infected SPF chickens than in infected conventional chickens.

The occurrence and the number of colonies of C. perfringens

was higher in SPF 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

conventionalizedd" chickens), the mortality rate was high

when C. perfringens and S. fecalis were members of the








microflora. A possible 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 (1969) 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 newborn

piglets due to C. perfringens type C infection (Field and

Gibson, 1955; Barnes and Moon, 1964; Bergeland, 1965).

In conventional chickens the lesions and reduction
in hematocrit values as a result of E. tenella infection

were more noticeable than in SPF 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 failure 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, 1963). 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 known.

Another feature of cecal coccidiosis, unrestricted
hemorrhage with no coagulation, was seldom seen in chickens

harboring only S. fecalis. Bacteria may have a definite









role in keeping the blood noncoagulated since poly-

contamination with C. perfringens and S. fecalis or C.

perfringens, E. coli, Bacteroides 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 when 1 day-old

chickens are exposed to E. tenella. Bacteria-, fungi-,

and PPLO-free chickens when exposed on the day of hatch

did not develop the pathognomonic lesions of coccidiosis

due to E. tenella. No mortality occurred in these groups.

However, when the inoculation was preceded by inoculation

with single or multiple species of bacteria, lesions de-

veloped. Mortality and bloody cecal contents were noted

only in the group polycontaminated with Bacteroides sp.,

E. coli, C. perfringens and S. fecalis. Groups mono-

contaminated 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 4 weeks

old. Rose (1967b), studying the excystation of E. tenella

oocysts, observed rapid and greater excystation in chicks








aged 4, 5 and 6 weeks and less successful excystation in

1 day- or 1 week-old chickens. The greater suscepti-

bility of the 4, 5 and 6 week-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 low susceptibility of day-old

or week-old chickens was likewise ascribed to the less

successful excystation of oocysts as a consequence of the

less powerful 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, 1942; Ikeda, 1955, 1956, 1960; Hibbert et al.,

1969). Hibbert et al. (1969) found that oocysts of many

species of Eimeria, 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 walls of the sporu-

lated oocysts expelled in the feces of exposed chickens

was observed by Lotze 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









a result of rapid and greater rate of excystation seems to

occur in day-old chickens harboring certain bacterial

species which are also regularly isolated from ceca of

chickens showing typical lesions of cecal coccidiosis

while 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 was harbored in the ceca, if

the rate and degree of excystation is correlated with the

development of the disease. In the newly-hatched conven-

tional chicken the only microorganisms predominant were

S. fecalis and E. coli. It was also found that the patho-

logical manifestations of coccidiosis were 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 known and has not been studied.

Normal excystation and subsequent liberation of

sporozoites may not be the sole process which will in-

fluence the development of pathological manifestations

in E. tenella infection. Horton-Smith et al. (1963)

reported normal excystation of oocysts and penetration

of liberated sporozoites in immune chickens. It is also

known that much of the pathology in cecal coccidiosis is

due to the maturation of enormous numbers of the large 2nd

generation schizonts which destroy the epithelial cells,









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 cocci-

diosis originally described by Tyzzer (1929) and Tyzzer

et al. (1932) failed to develop in chickens raised

bacteria-, fungi- and PPLO-free. No clinical symptoms,

mortality or decrease in hematocrit values following ex-

posure with standard doses of sporulated oocysts were

observed. Likewise, no visible lesions were noted in

these chickens. Chickens raised simultaneously under SPF

and conventional environments and inoculated with the same

standard dose of sporulated oocysts exhibited the classi-

cal cecal coccidiosis syndrome.

There was also a marked difference between bacteria-

free and SPF or conventional chickens based on histopatho-

logic findings. Ceca of the SPF and conventional chickens

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 was seen to be stripped down to the base of

the submucosa. Large 2nd generation schizonts lying mostly

free and a few within their host cells were the predominant

endogenous stage seen in such cases. But in the ceca of









bacteria-, fungi- and 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

was more or less intact and other tissues were undamaged;

no large 2nd generation schizonts were seen. A few en-

dogenous 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-,

fungi- and 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 patho-

logical manifestations are not developed. In fact, the

bacteria-, fungi- and 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 develop-

ment seems to have been retarded. Clark et al. (1962),

in their studies with bacteria-free chickens, observed a

12-15 hour delay in the appearance of 2nd generation mero-

zoites in the feces of bacteria-free chickens. However,

they found no difference in the course of infection in









bacteria-free and conventional chickens. The exact

experimental procedures and results are not completely

known to compare their results and those from the present

study.

Wagenbach et al. (1966), describing a method for

sterilizing coccidian oocysts employing Clorox and sul-

furic 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 work been

given, it is not possible to compare their results with

the present work. Their work 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. Monnr and Honig (1954) reported that when

oocysts are treated with 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








eggs and oocysts has been found to be very effective (Doll
et al., 1963; Bradley et al., 1964; Hegde et al., 1969;
Springer et al., 1970). Under the light microscope, no
damage or changes to the oocyst wall were seen when pera-
cetic 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 (1966,
quoted by Hegde et al., 1969) who observed no mortality

in bacteria-free chicks when 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 de-
velopment 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 et al.,1959
Johnson, 1971). Springer et al. (1970) reported survival









of H. meleaRridis without disease production in gnotobio-

tic hosts. Confirming the synergistic role of certain

bacterial species (especially C. perfringens) and H.

meleagridis in the production of infectious enterohepati-

tis in turkeys reported by Bradley et al. (1964), 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 entero-

hepatitis.

In E. tenella infections, the exact role of the
bacterial species is not known, 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 et al. (1967) and

Rohovsky and Griesemer (1967) found feline infectious

enteritis in the germ-free cat to be a mild, nonfatal

disease consisting of leukopenia, thymic atrophy and

lymphoid depletion but without 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 con-
tain the major portion of the gastrointestinal flora

(Timms, 1968), E. tenella may have developed an apparent

obligate relationship with certain members of this micro-

flora for pathogenesis to commence and this relationship
would have influenced the overall host-parasite relationship.









The contribution of indigenous bacteria of the

cecal pouches in producing cecal coccidiosis syndrome

was first suspected by Ott (1937). Mann (1947) reported

the bacteriology of cecal coccidiosis to be similar to

that of "six-day disease" incited by C. perfringens,

enterococci and coliforms, and characterized by occlu-

sion of ceca with a core and hemorrhage. Johansson and

Sarles (1948) also reported a possible involvement of

E. coli in the etiology of cecal coccidiosis. Their

study and the present study showed 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 seen

in day-old chickens may also be due to the same reason.














SUMMARY AND CONCLUSIONS


Typical cecal coccidiosis did not develop in

bacteria-, fungi- and PPLO-free chickens inoculated with

a standard dose of infective oocysts of E. tenella.

However, small numbers of E. tenella were able to develop

at a slower rate in bacteria-, fungi- and PPLO-free

chickens as compared with the rate seen in conventional

chickens. In chickens monocontaminated with C. perfrin-

gens, S. fecalis, E. coli, Bacteroides sp., Lactobacillus

sp., Mucor sp. or C. albicans, exposure to E. tenella

oocysts resulted in mild cecal coccidiosis. Cecal cocci-

diosis in chickens polycontaminated with bacteria was

comparable with that seen in conventional or SPF chickens.

Higher mortality but lower mean gross lesions due to

coccidiosis were seen in SPF 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), enterococci predominated in the ceca while

in older chickens (4 to 5 weeks of age), Lactobacillus sp.

predominated. Bacteroides sp., E. coli, Lactobacillus sp.

and S. fecalis were regularly isolated from conventional

chickens at 1, 2, 3, 4 and 5 weeks of age. Isolation of









C. perfringens was infrequent in conventional chickens,

but frequent in SPF chickens. Inoculation of chickens

with E. tenella resulted in stimulation of growth of

C. perfringens both in conventional and SPF 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.





























APPENDICES





























APPENDIX A




















cX0, 0.o O0o COo
orcvo orNrO \o-Io ci4no
o-4c N-4 CM we


P4
0
0
,-1

po
0
Od E




+ 00
0
P40
PfE






0
CO
o









m .
S0
0O
o^

0 0
c *
o 0
ft^
Bo


0



Hr-IH
F4H
ao










09
OH











oS
0

o






,0
Or

0 4-



0

0
Ol



0


m
O
0
0 0
SOO0
0 00
00


rA r -- -- V- U"
T-4 CM CM m


'-4,l O
enw w


m M a

O 0 O
S0 -40 0
00 00 00

cr4 o I 0 40-
.00 0.00 OO
00 4400 q-4


ax!(P ai !M

co
I'

***


a,
rd










bro
c
ac


to
CO
0

000
o0, 0
0 0 Q)
p; aSL
0a4)
CC 4-)


m

0
O0
0 0
,-I 0 0
0C- 0)0
OOfJ
odesf


Ucor\ *-1, ,D- -4t-4H10o -4-Co0
%00 0o CD U"\V,-I 4 00o
--t-t V-4V-1 \ --.t \o n^















110 nc
o00

CNI VA
CcI-\
** *


C 0o0 *>- *
NOO D
NW *N* t


m
0
%-4F



O 0





,oV





00
0d








0
o4
OX











00
Pmc







rd
4a)
403 0








00
o







rk

0
OH
o o
00


M














S0


P4
kO
OO
1. 0
D) 0







0~0
0-i
Smd









OH







co
Qm
00









P4
cO
4 0
(D0


m< 0











o rd
oo
C H


m 0






ft
o co
O H
t t i


t *N c
4-V~ C'
v~ryS~


"Crm
0\C%
M %-I


o* H






HO 0 p

00 0

oil 0 mi d


\% .
c'-ts.s.


CO Cr\
N r-4 Z


4
t!
p* m
m m


o H H


0 O H 00
04 0
0 0
*cc










I- '
i-4 CM


m

-H
P






0
0i

4

t to
z 0}
m ) H



r-4-i O .,-
r-4 OI) o0
O OH I Hi 0 o
0 Ol &i- O !2 0
0 0 0 1 0 0
Ci-4o 0 J4-1 P4. 0
0003
cf
wMIl Mlwjm li4 :


-4l LI-












P,
N 0
00
4-4 o)-:t co -t o
O rd nro q 1o .
"-i cc

o !-1 a
0 r-I




d d m
< a mm a
0 H m M r- O az
P4 r- C m i .-
m *-H r -I r-i d rd
(D I 4 a ri 4i a 4. .
0 0P p r-I r0 O PN
*H c- 000 000) l
0 r-I 01G P "-,
O 0 00
P4 O cc rd


r' 4




O.*
02 A 0
9 A 4 0


Ho H H
H0 0 H d 0- .

NI 'r C C4 -t T-
-P 04 HO
H 0J0 0 040







m m moom a
00



-H r-4 0 0

cd rd 0 r-I iC a-4 4-I 0 'd
0 me W* c CO H 0 p4 0
0 -1 O 0) P0 N


a 4--1 a 4- 0
4' 0 0P 0 t0 0






Sco 0 0
H O < H0 <

-ro
rzI 02 02 020002 0






00 0
0 0 4 H C-










Q) O,
O1CO#

to rd 4 c C rd
04a4 0 ak 0 0



cC m ) am : 3
4' W bo 4) 4.4 M d- W 0 es-i 0
043,. 0 bOD 0- 0o b4O -
M m>M H m>MO H
Wo *bd c0?-p o *U-4 c*4
04,F00O4) 040 b
S0 0,d41 4 0 4d 41P
E memo (l) emeo riSr o( aor


41


00

s CC


H 0
4
P:4


*d





1H
-m4








41
14

00


4,0
Idd



ei 0
94 &
I04






m i









0
43
o
0 14
0











P4)
01 f
i-t <





















pt


oa -
P0 m

oM w
O m

0

o a
o bDO
0 dri m




41-
94
q1-











'-I



-H




Ca,
0
s




a,








0 4f.X: *

"4.3-P r,
pq L S-P


04 )1






0-) F 0 tg a) }-
CC F:44'

s 0 0 0



S4) O F4
goegagPO


41-


Co

Zm P
0


oH i -I
r BO d d
o ao (


r-I




0 med 0


:4 m a





+
+
+r


o
0


H m Id rd
.-I .,.4


0 -H H'd 0
Sam P :


0C
a
X44
00
.-














00 oI

>oo rm O B 3Eam e 0Oa
d0 0 d 0 H m m cc ,D



s 0> ( e (U hO 0 4-N x0 m M0 )
0d I (DOt) 00F! H 0a0) 020

0i- Mh o h Et.,-bO gH a to ta o
60 M P D0b Ov bOO O
CH bD c efl4 0 .cPi-I V ;4 a4d O q r4
.w w >W>-' P M r M.0 m > e0m > 0 m


O




0
o 91 0







O 0






















r-




H
0 ac-o

















0
ftO

































Fm.
IL cd
Om 0

m00 Wq
r4l
03


















1-1








p Scir
B2; (0 i-







k)0


rr







0
Zi



4,
H-l 0
0


3 ri
0


H- 0,'d

o H H
hmb


ad
()
4-)
H 0
o

do
*H rh4

H mi


o o


0)
H O0
d b
o

og


o -H H
a m ,


rd


5g a
rl 0


H m 'd
So p


+
+


H
.1-I





a





fod
Sm0p

OH












+


4 .

-?* M -a


-^ vr \o


0 0 0















A 0 a)
O
0 --
0 4 4-.

Stom

on
PON


Ao0
0

0 aoF:









Hd
ca













o
Cl




H
0










0









Sa)
5003









0 H





*H
i-H












E-1
0:4 4


0






















a





0


kmi
Od r


(D a
0 0
uOH









I
I


43
cc









0
I-I


H^


H i


a1) 0
H ho


43 H
0

-H 0
mI m m
0l 0
0

S0

S0) P
o 4 0
0 m 0

o 0 4

o 0a 0






'c P4 I
1 4) 4-
0 0
4a P 4 0


o o 10




* P4 0
0 43 0 o3
o 0 0 0





cc 0 P
o P4



o 0- 0 -











94 t o Po
0$ 0
o a)
o m C)
o 6- m a s
H 4 0 o


0 a c A
-0 U0


H aZ 4) HP
a, a6- 4
+ 0 0




oi ZQ 4)


- 4I O Q C0
+ o a F
+ 0 4 4
0 O l OH



a0 ) *
0 0 0 B






m a


0
9-
P
0
m
0
a)
m

0
i-4


a)


0
tkl
0

4-
0


41-


0
4

$4


ca

0

a)
Fi






4
0


e4
0
c4



0




Ua
0


0
1--4





a*




00
m1m
0
m4

4 ,


-H
ch
13
0.










H-












4-4 a,
0
mQ
F :4 m *

r a, aip
a) M 0 42 a,
o :'d a) a
0 m a) m
m 60 O h 0 4

- b 0 b0 0 a,
0' -acdrd
0 CO r 4 0 CC
o0 (1l M Zid


a, 0

cd rd
rdH a,

0 4,3
ca *a 0

E-4 >-
E*- m fr


ri
m



o
4-

0
(DO
4-1

a, H
A -l

O r--
ao
H "-I






rd i

8 d




a) m
a














0 03


04
o








m d
0)
4P



a,
+4
03
cut


O)
*HI-4 S
o0 mh
O *Ri-4





EO 4 c
:4'd cc







o bo
mi- i
o m

0 w0
o hc







Io r-I

oa,




H

ow

r-1 -
H-H


h0

O


a,
:c *a
B 0 iV-




4 41
xad a
rA A 4-)


m





E-4
00







E-1


*



a
o

o
o


o

a,
N
r-t
.r4

a,
4i-)




4
0

o

0
cc














I-L
0
m

o
0











4.4







4-
0




o
r-











O
af








0



.-I
Sr







+:


CO







0







+