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This is to certify that the
thesis entitled
Coocidiosis in Pheasants

Reared in Confinement

presented by

Julie Ann Smith

has been accepted towards fulfillment
of the requirements for

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MSU

 

 

 

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LIBRARIE

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COCCIDIOSIS IN PHEASANTS RBARED IR OONPINEHBNT

BY
Julie Ann Smith

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Medical Technology Program

1986

ABSTRACT

COCCIDIOSIS IR PHRASANTS REARED IN CONPINEHENT

BY
Julie Ann Smith

The epidemiology and pathology of coccidiosis in game
farm reared pheasants were studied over a nine-month period.
Three hatches, each reared during a different season (spring,
summer and fall), were divided into two groups, one receiving
a coccidiostat (control group) and one without (study group).
Every week four birds from each group were randomly selected,
killed, weighed, and examined for coccidial species
identification. Four species were identified: aimggia
d_u_L__aLi_§den , E. mm. a. Begins. and E. 931212121.
mm dugdgnalis was seen first following winter but 5.-
phgsiani appeared most frequently and was often found with a.
pacifiga. Eimgria gglghigi, the most pathogenic species, was
seen only on occasion, primarily in the study group.
Although severe coccidiosis did occur, increases in mortality
tended to be stress-related, i.e. during movement to a new
unit. Susceptibility to disease was more apparent in younger

and non-medicated birds.

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude and appreciation
to members of my committee: Dr. R. Flint Taylor, my research
advisor, for allowing me the opportunity to explore new areas
within the scientific community and myself; Dr. Tjaart
Schillhorn vanVeen, for setting expectations that compelled
me to search deeper and longer; and Ms. Martha Thomas, my
academic advisor, teacher and friend, for believing in me,
supporting me and guiding me throughout my graduate program.
I am indebted to the DNR and its employees at Dansville, in
particular Martin Pollack, who gave so graciously of their
time and selves making this a truly coordinated effort.

It is with affection and gratitude that I thank Susan
deGraaf, animal research technician, for sharing her
expertise in parasitological techniques as well as her sense
of humor. I am also grateful to Mae Sunderlin for teaching
me histological techniques and to Alma Shearer Geany for
sharing her equipment, instruments and time.

The support and friendship of my fellow graduate
students, in particular Donna Duberg, have been deeply

appreciated. Their encouragement, willingness to share and

ii

honesty have greatly contributed to the value of my graduate
school experience.

I would like to express a very special thanks to my
parents for once again lovingly offering their support as I
pursued an educational goal. A special thanks also goes to
Chester P. Callahan who by caring provided me the final
impetus and strength for completion of my program.

Without the willingness of my daughters, Charlotte J.
Smith and Janeen L. Smith, to change their lifestyle and
sacrifice the security they had known so that I could return
to college, my graduate degree would be only a dream. It is
with love for them and appreciation for their patience and

support that I acknowledge my deepest debt of gratitude.

iii

TABLE OF CONTENTS

List of Tables. . . . . . . .
List of Figures . . . . . . .
List of Plates. . . . . . . .

INTRODUCTION. 0 C O O O O O 0
LITERATURE REVIEW . . . . . .

Introduction . . . . . .
Life Cycle . . . . .

The Parasite in the External
The Host-Parasite Relationship . .

Pathology. . . .

Eimeria sp. of the Pheasant.

Pathology of Coccidiosis
Oocyst Counts. . . . . .
Preventative Therapy . .
Epidemiology . . . . . .

MATERIALS AND METHODS . . .
General Considerations
Experimental Pens. . .
Selected Hatches .
Histopathology .
Parasitology . .-
Epidemiology . .

RESULTS . . . .

Coccidial Species. . . .
Oocyst Counts. . . . . .
General Performance. . .
Gross Lesions. . . . . .
Histopathology . . . . .
Epidemiology . . . . . .
DISCUSSION. . . . . .

Coccidial Species. . . .

Effects of Infection on the Pheasants.

in Pheasants

Environment

The Pheasants in the North-D Pen .

sumary O I O O I O O O 0

iv

TABLE OF CONTENTS, CONT'D.

RECOMMENDATIONS . . . . . .
APPENDIX. . . . . . . . .

LIST OF REFERENCES. . . . .

tthI-J

LIST OF TABLES

Description of Eimeria Oocysts.

Dimensions of Indoor Units and Special Pens

Mean Dimensions of Oocysts.

Mean Temperatures During Confinement to

North-D Pen . .

Frequency of Coccidial Infections in Birds 8

Weeks or Less .

vi

23
34
39

54

55

LIST OF FIGURES

Sketch of DNR Pheasant Rearing Facilities
at Dansville, Michigan. . . . . . . . . . . .

Life Cycle of the Coccidia, Eimggia sp. . . .

% Mortality and Oocyst Count for Each Week
of a Hatch, Both Groups . . . . . . . . . . .

Mean Weight of Birds (grams) as Measured per
week Of HatCh 0 O O O O O O O O O O O O O O 0

viii

P39;

42

45

Plate
la

1b

1c

1d

LIST OF PLATES

Photomicrograph of a transverse section of

the upper ileum showing Eimggia phagiani in

various stages of development within the tip

of a villus. 10X. . . . . . . . . . . . . . . . 49

Photomicrograph of a transverse section of

the jejunum showing Eimggia phagiani lining
the sides of a villus to the base, various

Stages. 40x. 0 O O O O O O O O O O O O O C O O O 49

Photomicrograph of a transverse section of

the caecum showing 31m_;1§ cglc chic ci in the
glands. 10X. . . . . . . . . 51

Photomicrograph of a transverse section of

the caecum showing Eimggia cglchici in a
gland. 40x . . . . . . . . . . . . . 51

viii

INTRODUCTION

Each year several thousand pheasants are hatched, reared
and released by the Michigan Department of Natural Resources
(DNR) as part of a program known as "put and take“. This
project was initiated as a means of providing sportsmen with
an adequate supply of pheasants following a significant
decline in the wild pheasant population. Changes in land—use
patterns in southern Michigan during the 1960's reduced
available feed, dried up marshes required for winter
protection and eliminated many nesting areas through early
cutting of alfalfa fields. Because of a lack of living space_
for the pheasants, stocking the countryside with eggs and
chicks would have done little to increase the numbers of
birds surviving to maturity. Thus a decision was made to
breed and raise the birds in captivity and release them at
18-22 weeks of age. In 1972, breeder pheasants were placed
at the Mason Game Farm and by 1973 the first pen-reared birds
were released at selected Put-Take Hunting Areas around
southern Michigan.

The facilities for rearing the pheasants are presently
located in Dansville, Michigan. They include two buildings
with special brooder units where the birds are kept from one
day until 12-weeks of age and range pens for adaptation to

outdoor living (Figure 1). These fenced outdoor paddocks are

Figure 1: Sketch of DNR Pheasant Rearing Facilities at

Dansville, Michigan

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4
situated on a slope that begins its incline a short distance
from the rear of the indoor facilities. During rainy periods
water flows down the slope collecting in those pens closest
to the buildings where pheasants 11-13 weeks old are kept
while adjusting to the outdoor environment.

Many of these younger pheasants die when the rains are
severe, accounting for a loss of approximately 10% of the
total flock. On the basis of clinical symptoms, macroscopic
lesions and the observation of oocysts, DNR veterinarians
have diagnosed coccidiosis as the cause of death.
Coccidiosis is a parasitic disease in which tissue cells are
invaded and destroyed by specific protozoan organisms called
coccidia.

Although pheasants reared exclusively in the wild are
also susceptible to coccidiosis (Norton, 1967b), the
potential for a lethal infection increases among those birds
reared in a close environment (Fayer, 1980). This is due to
a higher concentration of infective parasites per unit of
area and subjection of the birds to stress-producimg
situations which, if severe, may exacerbate a preexisting
coccidial infection.

Pheasant mortality and weight loss can usually be
minimized with the use of appropriate anti-coccidial drugs
and the application of good management techniques such as
those used at the Dansville facilities (see section on

Material and Methods and the appendix). However, even the

5
most rigid adherence to existing management policies may not
prevent serious outbreaks of the disease if conditions
develop which are beyond the control of the management
program.

The pheasant mortality rate at Dansville attributable to
coccidiosis is an economic burden of concern to the DNR. The
purpose of this investigation is to help in the development
of a new management program which will effectively control
the disease thereby reducing the coccidial-related deaths.

The specific objectives include determining (l) the
identity of those coccidial species involved and the pheasant
age group affected by each, (2) the species affecting the
animals during transfer to outside paddocks and (3) the
influence and severity of infection level at various ages and

the effect on the performance of the birds.

LITERATURE REVIEW

Intrgduction

Because poultry are of significant economic value,
scientists and poultry breeders have, for many years, had an
intense interest in any factors that affect the numbers and
quality of birds and eggs produced for market. Poultry, when
reared in close confinement, are at risk to a variety of
disease conditions that can (1) increase the mortality rate,
(2) reduce the weight gain, (3) alter the bird's general
appearance, and (4) affect the quantity and quality of eggs
laid. Coccidiosis is one of the more serious and common of
these diseases.

Research on coccidiosis in chickens has yielded a vast
amount of information on the parasite, its relationship with
a host, the influence of environmental factors and modes of
controlling the disease (see reviews by Horton-Smith, 1949;
Farr and Wehr, 1949; Edgar, 1955; Horton-Smith and Long,
1959; Ikeda, 1959; Rose and Long, 1962; Krassner, 1963; Long
and Horton-Smith, 1968; Vetterling, 1976). Much of this
informational data on coccidiosis in chickens can be applied
towards a general understanding of the disease in pheasants.
Information relating specifically to the epidemiology and
pathogenicity of coccidiosis in the pheasant is scant.

6

7
Instead the focus has been primarily on identification of

species and description of the oocysts.

Life Cycle

The coccidia included in the genus fiimggia have a life
cycle divided into an endogenous phase and an exogenous phase
(see Figure 2). The endogenous phase includes reproduction
by asexual (schizogany) and sexual (gametogony) processes, a
characteristic of most sporozoa. Sporozoites, the infective
form of these coccidia, penetrate a host cell, round up to
form a mononuclear schizont and begin nuclear division. As
the number of nuclei increases so does the cytoplasmic mass
until a mature schizont with many nuclei is produced. The
cytoplasm envelops each nucleus then breaks off, yielding
small, elongated mononuclear formations called merozoites.
The number of merozoites is equal to the number of nuclei
within the mature schizont. Their release from the host cell
into the intestinal lumen represents the completion of one
asexual generation. The total number of asexual generations
to be completed is a characteristic of the species, but most
often with those species specific for the pheasant, the
number is three. Free merozoites penetrate intact host
cells and then proceed to develop in one of two ways: (1)
they begin another asexual phase, with nuclear division and
subsequent formation of mature schizonts or (2) they begin
the sexual phase by maturing into multinucleated

microgametocytes and mononucleate macrogametes. Each nucleus

Figure 2: Life Cycle of the Coccidia, fiimgria sp.

LIFE CYCLE OF THE COCCIDIA

@6 Asexuol Re production %G\
a?

Excysmfion ENDOGENOUS PHASE
Sexual Reproduction
0 3/6
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EXOGENOUS PHASE

10
of the ndcrogametocyte yields one motile microgamete which,
upon release, may fertilize a macrogamete. After
fertilization, a wall is built around the zygote from
granules within the macrogamete and the structure thus formed
is the noninfective oocyst. Before an oocyst becomes
infective it must leave the host and enter the external
environment where the exogenous phase commences. Division of
the zygote nucleus is followed by the formation of
protrusions on the zygote surface. These grow and separate
into sporoblasts which go through morphological and
biochemical changes producing sporocysts encased by a
delicate wall. Division of the sporocyst nucleus and
cytoplasm forms sporozoites within each sporocyst. Coccidia
of the genus EM produce four sporocysts each with two
sporozoites. Once the sporozoites have formed, the oocyst is
infective, requiring ingestion and excystation for

continuation of the cycle (Kheysin, 1972).

The ability of ELEQLLQ oocysts to sporulate and survive
within their environment outside of the host is dependent on
certain conditions: (1) free access to oxygen, (2) a
suitable temperature and (3) sufficient moisture (Kheysin,
1972).

Oxygen is a requirement for the initial and final phases
of sporulation. In its presence amylopectin, a starch stored

in granules, is converted to the energy necessary for nuclear

ll

division. Development between these phases can continue
under anaerobic conditions, but sporozoites will never be
formed. An oocyst can survive an oxygen deficit for a period
of time that differs for each species. If it is not returned
to the aerated environment within that time, the oocyst will
suffer irreversible damage. Ina the field, oocysts are most
apt to be subjected to a lack of oxygen when contained in
material heavily contaminated with bacteria which rapidly
utilize available oxygen for their own respiration.

Oocysts exposed to temperatures in the range of 10-30°C
will sporulate normally and survive for periods of time that
potentially could span several months. Deviations from this
range in either direction will begin to have deleterious
effects cum cell division (Marquardt, 1960). The exact
temperature required before death of the parasite occurs is
determined by the degree of resistance inherent within the
species. Most oocysts are better able to withstand low
temperature extremes than high. Farr and Wehr (1949) showed
that some of the oocysts of E.- Lgpgua, E. maxim; and E.
acgrvulina, all coccidia of the chicken, could remain viable
during the winter when exposed to temperatures as low as-
19°C while Marquardt (1960) found that temperatures of 37°C
and 39°C caused permanent damage to both unsporulated and
sporulating oocysts.

Adequate moisture is required by the oocyst in order to

maintain the integrity of the cell wall. As water is lost

12

the wall wdll collapse pressing against the zygote and
inhibiting its development. A humidity level of 90% or
higher is considered adequate (Horton-Smith, 1947). Below
this level deformation of the oocyst is also influenced by
temperature. As the humidity decreases and the temperature
increases, the percent of deformed oocysts increases (Ellis,
1938). Deformation does not necessarily indicate loss of
viability. If exposure to drying has been of a short
duration sporogony may continue once sufficient moisture is
provided. However, long term dehydration is highly lethal to
the oocysts. In terms of field and pen conditions, coccidia
tend to sporulate rapidly and survive for long periods of
time around water troughs, in moist soil and in litter with a
high water content (Hawkins, 1950).

Oocysts have proven extremely resistant to a variety of
chemicals. This resistance is provided by the inner layer of
the cell wall which is composed primarily of proteins and a
mixture of waxes (Ryley, 1973). The outer layer serves to
safeguard the oocyst from mechanical damage. Removal of this
external layer by sodium hypochlorite or distortion of the
cell with hypertonic salt solutions does not impair via-
bility. Chemicals lethal to the oocyst must be able to
penetrate the waxes by dissolution or alteration of
conformation. Among those capable of doing this are ammonia
(Horton-Smith, et a1, 1940), mercuric chloride (Marquardt, et

a1, 1960) carbon dioxide, methyl alcohol, various organic

13
reagents and highly concentrated bactericidal agents, e.g.

formalin (Kheysin, 1972).

s - a t ns

ZBoth initiation and continuation of coccidiosis, which
is a self-limiting disease, require ingestion of sporulated
oocysts followed by excystation, a two-phase process yielding
free sporozoites. Hibbert and Hammond (1968) suggested that
each species of coccidia has its own pattern of excystation
which has contributed to the adaption of the parasite to its
particular host. The purpose of each phase is to alter the
permeability of the oocyst or sporocyst wall. The first
phase is stimulated by (1) carbon dioxide, (2) a mechanical
action, or (3) enzymes, while a trypsin-bile mixture is
essential for the second phase (Nyberg and Hammond, 1964).
Among different hosts, the sequence of sporocyst and
sporozoite release varies. In gallinaceous birds, sporocysts
are released first followed by liberation of the sporozoites.
The two phases usually occur in different locations along the
alimentary canal.

Most coccidia will parasitize the digestive tract of
either a vertebrate or invertebrate host. A few, though, are
known to carry out the endogenous stage of their life cycle
in other organs, e.g. E, stigga in the liver of the rabbit
and E. truncata in the kidney of geese. Free sporozoites, of
pheasant and other gallinaceous bird coccidia, are genetical-

ly directed to invade a particular portion of the digestive

l4

tract. This predilection is based on the nutritional
requirements of the parasite, the physiological status of the
cell and the host's immune response (Vetterling, 1976; Long,
1971). .A very delicate balance exists between parasite and
host, wherein the degree of cellular damage by the coccidia
and the defensive activity of the host are in equilibrium.
Any change in the host's nutrition, its protective mechanism
or demands of the parasite will upset the balance resulting
in either increased damage to the host or interference with
development of the parasite.

Certain of the coccidia's nutritional requirements, such
as proteins, nucleic acids, glucose or vitamin 312, are
synthesized by the host or its natural intestinal flora,
while others are acquired via the host's diet. Researchers
have determined that Eimgria species require the dietary
vitamins thiamine, riboflavin, biotin, nicotinic acid,
pyridoxine and para Amino benzoic acid (pAB). Each is
essential for a specific stage of coccidial development, e.g.
Warren (1968) found that biotin is required for the first
schizogeny and nicotinic acid for second schizogeny by E.
tgngll . Removal of any of these vitamins whether by
antagonism, inhibition or frank absence will arrest
development of a coccidium at that stage for which the
vitamin is a requirement.

Immunity is the host's most obvious protective

mechanism. Both antibodies and cellular elements constitute

15

the immune response. The presence of humoral antibodies is
an established fact, but research has indicated that
coproantibodies may also play an important immunological
role. These antibodies are of the IgA class formed in the
digestive mucosa. They represent a local defense and work in
conjunction with serum antibodies, which are brought into
direct contact with the parasites by an increase in gut
permeability (Rose and Long, 1969), immobilizing or
destroying either the sporozoites or one of the other stages
of development. At one time it was assumed that immunity was
exclusively local in nature rather than humoral, with the
tissue cell itself imparting a resistance to invasion by
sporozoites or merozoites. The cellular elements now
referred to when discussing immunity include macophages,
lymphocytes and granulocytes. The precise role of each in
coccidial immunity has not been definitely determined, but it
has been suggested that their modes of action are similar to
that in humans.

‘Within 24-48 hours following ingestion of a coccidial
dose by an immune host, irreversible damage has been
inflicted on the developing parasite (Leathem and Burns,
1967). Degree of immunity (total or partial) as well as the
nature of the coccidial species may influence which
developmental stage is affected. Rose (1968) suggests that
total immunity may inhibit the formation of oocysts while

partial immunity reduces the total number of oocysts

16

produced. Immunity is not always stimulated in the host at
the first introduction to the parasite. Assuming a moderate
number of oocysts per dose, the number of immunizing doses
required before the host is protected against a challenge
dose depends on the particular species of coccidia, e.g. E.
maxim; requires only a single dose in order to elicit a
response whereas E. alabgmgnsis requires four doses. This
characteristic, referred to as immunogenicity, is not only a
function of the number of oocysts per dose but also the
location of the coccidia within the tissue. Sporozoites
often localize in the epithelial cells lining either the
distal or proximal half of the intestinal villi, but they may
travel the lamina propria, possibly via macrophages, to
settle at the base of the villi or in the glands. According
to Tyzzer (1929) those species that develop superficially in
the intestinal epithelium require more immunizing doses than
those that develop deeper in the mucosa.

Immunity is usually specific for that species which
initiated it. Although cross-reactivity rarely occurs in
poultry, a host may be resistant to more than one species
provided a sufficiently large dose of oocysts from each
species has been ingested. Duration of immunity is dependent
on such factors as the mode of immunization, age of host and
the time interval between an immunizing dose and a challenge
dose (Rose, 1973). In birds not subject to reimmunization,

duration varies with the coccidial species. An immunity

17
stimulated by E, maxima is short-lived but one stimulated by
E- tenella may last several months (Long, 1962; Leathem and
Burns, 1967). Continual ingestion of infective oocysts may
provide the host with sufficient stimulus to maintain
indefinitely a competent immune status.

Coccidial survival can be enhanced by suppression of the
host's immune system. Fmeexisting infection with bacteria,
viruses or other parasites may render a host incapable of
responding immunologically to invasion by the coccidia while
stress-producing situations, metabolic changes or production
of interferon may potentially result in reduced effectiveness
or actual loss of a previously developed immunity. 0n the
other hand, immunity to non-coccidial antigens may be
suppressed by an established coccidial infection. Kaye, et
a1 (1965) superimposed a Salmgnglla typhimurium infection on
mice already infected with Elagmggigm peggngi. These animals
were compared with mice infected with only one of the two
microorganisms. Higher mortality occurred in mice with the
superimposed infection which the author attributed to
salmonellosis rather than the parasitic disease. Coccidial
infected hosts, although presenting a severe immunosup-
pression against unrelated antigens, display high levels of
specific serum immunoglobulins (Krettli and Bereira, 1981).
Orjih and Nussenzweig (1979) demonstrated that reinfection by
the original organism did not abolish an established

immunity. Animals repeatedly infected with blood parasites

18
and immunized with irradiated sporozoites had unaltered
antibody titers despite high levels of the parasite.

The influence of host age on coccidial survival differs
with the conditions under which the bird was reared. An
older host that has been housed in a coccidia-free
environment will be more susceptible to both parasite
reproduction and pathogenic effects than a young host reared
similarly. Long and Horton-Smith (1968) and Krassner (1961)
have suggested that this may be a result within the older
bird of better parasite excystation, increased numbers of
intestinal cells and physiological conditions more conducive
to sporozoite penetration. However, young birds are far more
vulnerable to disease than older birds when both age groups
have been reared under conditions that allow for the
development of immunity (Horton-Smith, 1947). Thus, the
coccidia are better able to survive in young chicks and non-
immunized older birds.

Investigators have shown that the size of an ingested
dose is directly related to the effects produced within the
host (Long and Horton-Smith, 1968), but inversely related to
the yield per infecting oocyst (Krassner, 1963). The
exception to this occurs with what is known as the "crowding
factor“ in which both pathogenic effects and yield are
diminished per infective unit when an extremely large dose of
oocysts is ingested. A few possible explanations for the

occurrence of this phenomenon have been proposed: (1) very

19
rapid development of an immune response (Fayer, 1980),
(2) loss of healthy tissue for continued reproduction
(Krassner, 1963), (3) possible production of interferon or an
interferon-like substance which inhibits normal replication

(Long and Milne, 1971) and (4) competition for nutrients.

warm

Evaluation of a bird's performance and general health
status may reveal symptoms that indicate disease. The
clinical signs suggestive of coccidiosis include myasthenia,
lethargy, weight loss, poor feather growth and diarrhea.
These are related to physiological and biochemical changes
such as reduced muscular glycogen stores (Long, 1973), blood
loss, anorexia, vitamin deficiency induced by poor absorption
of nutrients (Briggs, 1946; Preston-Mafham and Sykes, 1970)
and malabsorption due to loss of intestinal epithelium
(Preston-Mafhan and Sykes, 1970). The absence of clinical
symptoms, however, does not exclude the presence of
coccidiosis within the birds. The disease may be subclinical
and undiagnosable until the intestines are examined
macroscopically and microscopically.

Lesions characteristic of diseases or conditions that
may have clinical signs similar to those of coccidiosis, e.g.
vitamin deficiency and wood-chip blockage, may be revealed by
examination of the mouth and crop. Wood-chip blockage is
common among very young chicks 2-3 days old. Litter is

ingested in quantities which pack the crop. The sensation of

20
fullness in the stomach which ensues decreases the intake of
food while the blockage in the crop prevents what little food
is ingested from reaching the gut. The chick subsequently
dies from starvation.

Macrosc0pic examination of the gut will reveal lesions
produced by physiological changes within tissues infected
with the coccidia. Blood vessels become enlarged following
an increase in blood flow (hyperemia) induced by the immune
response to deliver granulocytic leukocytes and humoral
antibodies to the area of infection. Leakage of protein into
the mucosa with the subsequent development of edema (Long,
1973) produces a thickening of the intestinal wall. The
proteins are lost due to changes in the mucosal blood
capillaries. Proteins are also leaked into the intestinal
lumen where fluids increase to dilute the proteins and mucus
accumulates from destroyed glandular cells. Together they
produce watery intestinal contents. Hemorrhage will add to
the fluidity of the material within the intestines. It is
observed on gross examination by redness of the serosal
surface or areas of petechiae.

According to Long (1973) a change in body weight of an
animal is considered by many workers to be the only indicator
of subacute coccidiosis. Weight-gain reduction begins on the
4th day of an infection and reaches its maximum by day 5 when
both tissue and oocysts are being sloughed. PTeston-Mafham

and Sykes (1970) related weight loss to severe depression in

21
the absorption of nutrients caused at least in part by

destruction of intestinal epithelia and to anorexia.

'm s s t

Ten Eimerig species have been described in pheasants:
Eimggig dispersa (Tyzzer, 1929), E. phasiani (Tyzzer, 1929),
E. lgmggrgni (Yakimoff and Matschoulsky, 1937), E. pggifiigg
(Ormsbee, 1939), E. W (Ormsbee, 1939), E.
ggnngguscus (Ray and Hiregaudar, 1959), E. 2.12;; (Bhatia,
1968), E. duodenalis (Norton, 1969a), E. cglchici (Norton,
1969b) and E, tetgrtooimig (Wacha, 1973). Only six of these
species have been found and confirmed in the ringneck
pheasant, Ehasigngs cglchicgs Eggguggus, the most common
pheasant in the United States. Three of the remaining four
species have been found in pheasants other than the ringneck
and the fourth species, E. digpgggg, is regarded by Pellerdy
(1974) as species incerta since it has been reported in a
variety of gallinaceous birds.

Identification of the coccidia is based on (1)
characteristics of the oocysts, (2) location of the different
developmental stages within the intestine and tissue, (3)
length of the prepatent and patent periods and (4) immunolog-
ical specificity (Joyner and Long, 1974). The use of more
than one criterion is particularly essential when the
observed oocysts exhibit characteristics similar to those of
another species. A mixed infection within a single host may

require the isolation of individual species for innoculation

22
of 'clean“ hosts. Table 1 describes the oocysts of six of

the Eimggig species found in the ringneck pheasant.

atho o o occ'dios s 8 nt

Gross or macroscopic lesions due to infections with E,
pacifica and E, megalostomgta are rarely detectable (Ormsbee,
1939). Kiwanis 291211.121. a. abasisai and E. Malia.
however, are capable of producing severe lesions as reported
by Norton (1967b, 1967a) and Trigg (1967b), respectively.

Attention to the location of these parasites both within
the cell and the tissue is of importance because of the
relationship between pathogenicity and site of development.
Tyzzer (1929) reported that the deeper the parasite
penetrates the tissue, the more severe are the effects on the
host. It has been suggested that this is due to a stronger
immune response. Parasites that locate above the nucleus of
epithelial cells lining the superficial villi usually do not
invade other areas of the tissue. Lesions are then confined
to destruction of surface epithelial cells, which are readily
replaced, and a mild to moderate cellular response.
Coccidial localization below the nucleus of an epithelial
cell is potentially followed by transversing of the basement
membrane and subsequent invasion of the lamina propria,
tunica propria, glands and submucosa. Disruption of
capillaries will cause some hemorrhage while invasion of
progenitor cells in the crypts of Lieberkuhn inhibit their

division which results in villus atrophy and subsequent

223

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24

reduction of surface area available for absorption. Invasion
of these different areas of the tissue by the coccidia
stimulates an influx of inflammatory cells and erythrocytes.
Lymphocytes appear late in an infection. In moderate to
severe infections, necrosis and fibrosis will develop,
usually following release of most of the oocysts into the
lumen.

Eimggig cglchici is the most pathogenic of the pheasant
coccidial species, responsible for caecal coccidiosis in
young chicks 2-4 weeks old. First and second generation
schizonts locate in the glands and lamina propria at the base
of the villi, usually below the nucleus (towards the basement
membrane) of the infected epithelial cell. The third
generation schizont is found in the caecal glands while the
gametocytes infect the epithelial cells lining the caecal
mucosa. A mortality of 100% occurs in birds dosed with
320,000 oocysts. Soft white cores of necrotic debris,
hyperemia throughout the intestines and a mucoid exudate are
produced with this number of ingested oocysts. Fewer oocysts
produce less severe lesions.

Eimerig pnggiggi is a moderately pathogenic species
producing 100% mortality with a dose of 500,000 oocysts. It
is most often a parasite of young pheasant poults. Location
of the first asexual generation is in the glands and at the
base of the villi of the upper one-third of the small

intestine and the second is in the epithelial cells lining

25

the villi of the upper half to two-thirds of the small
intestine. The third generation schozonts and gametocytes
are distributed throughout the small intestine and the
proximal half of the caeca. They too infect epithelial cells
lining the villi” iPosition within the epithelial cell is
usually below the nucleus. An edema, thickening of the
intestinal wall, hyperemia, some hemorrhage, infiltration of
many eosinophils into the tissues, and “ballooning“ of
epithelial cells characterizes disease with this species
(Trigg, 1967a).

A dose of 5,000,000 oocysts is required for 100%
mortality of 3-week old chicks infected with E. daggenalis, a
mildly pathogenic species. Norton (1967a) has suggested that
those deaths related to infection with E. dggdggglig may, in
part, be also due to a bacterial infection enhanced by the
stress of coccidiosis. Although 3-week old chicks were used
for pathogenicity studies, disease from this species is not
necessarily found predominantly in younger birds. E.
dggdgnalis locates in the epithelial cells lining the villi
of the duodenum and upper small intestine. Location within
the cell may be below the nucleus but usually is above
(towards the mucosal surface). A pinkish mucoid exudate
covering the duodenum and upper small intestine, caeca
distended with a yellow foamy fluid and edema are
characteristics lesions of more severe infections with this

species.

26
QQE¥§£_QQEEL§

A diagnosis of coccidiosis cannot be based solely on the
observation of oocysts in the feces (Davies, et a1, 1963).
The presence of coccidial developmental stages within the
intestinal tissue (Norcross and Washko, 1970) and the
occurrence of characteristic gross lesions are more
diagnostic since non-infective oocysts or oocysts infective
for another host may be ingested and directly eliminated.
Conversely, oocysts may be absent in the feces of hosts with
an infection severe enough to cause death (Davies, et a1,
1963). A lethal oocyst dose of a species whose most
destructive developmental stage is a part of the asexual
cycle may cause death to the host before oocysts have been
produced (Horton-Smith, 1947). Death may also follow the
completion of the endogenous phase if the intestinal
epithelia are so completely destroyed a malabsorption-type
syndrome develops. The value of the oocyst count lies then
with its use as a monitor of the progress and severity of an
established infection when only one species of coccidia is
involved and reinfection is prevented. The endogenous phase
of coccidial infection consists of two periods: (1) the
prepatent period, which begins when the first sporozoite
penetrates a host cell and which ends when the first oocyst
is eliminated into the external environment and (2) the
patent period, which extends from passage of the first oocyst

to passage of the final oocyst (Kheysin, 1972). Both periods

27
last several days, the exact number dependent on the
infecting species. As the patent period progresses, the
number of oocysts present in the feces will increase until a
peak is attained after which the numbers will decrease. The
size of an ingested dose will influence both the severity of
the disease and the number of oocysts eliminated. Thus the
oocyst count, if performed on a daily basis, can provide
meaningful data for evaluation of the disease. Under
conditions of intense rearing, however, it is impossible to
prevent reinfection and often more than one coccidial species
is present. The information garnered from the oocyst count
when mixed infections, reinfections and sequential infections
are present is limited. Its value is confined strictly to
numbers which, if significantly elevated, will indicate a
serious infection. Examination of the tissue will be

required for establishment of the disease progress.

2W

Anticoccidial drugs are applied to hosts in environments
where the immune system is unable by itself to provide
adequate protection against the development of coccidiosis.
Most act by interference with enzyme activity at one or more
of the developmental stages. Amprolium, for instance, acts
reversibly to inhibit thiamine (Cuckler, et a1, 1960)
affecting the first generation schizonts of E. adggggides in
turkeys (Warren and Ball, 1963) and second generation

schizonts of E. gcegyglina in chickens (Warren, 1968).

28
Monensin, an ionophore, inhibits the transport of cations
while arprinocid blocks hypoxanthine (Braunius, 1982).
Proper selection of and treatment with a particular drug
allows for development of immunity with no negative effect on
weight gain. Continued use of the drug may induce the
development of resistant coccidial strains. However, drug
tolerance, which Braunius (1982), describes as “a state of
insensitivity to a drug which ordinarily causes growth
inhibition or death of a cell," is more likely to occur with
long—time usage. Both resistance and tolerance are dependent
(M1 the Eimggig species involved and the number of passages
the species has undergone through a host. Switching to a new
drug may relieve problems incurred with resistance while
upping the dose may override the reduced response by a host
that has developed a tolerance.

Coccidiosis in pheasants has been controlled by the
addition of anticoccidial drugs to the feed or drinking
*water. Among those used in pheasants have been
sulfaquinoxaline, amprolium, zoalene and amprolium
hydrochloride/sulfaquinoxaline/ethopabate (ASE). No drugs
have been cleared by the Federal regulating agency for use in
small game such as pheasants. The cost of such a procedure
is not economically merited at this time. However, the
efficacy of drugs in pheasants has been tested through drug
trials by a few researchers (Norton, 1967a and 1967b; Trigg,
1967b; Jurkovic, et a1, 1982). Results obtained in the

29
1960's demonstrated the efficacy of amprolium against E.
gglchici and sulfaquinoxaline against E. W and E.
phasiagi. Zoalene was also found to be effective against the
latter two species but not to the same degree as the
sulfaquinoxaline. Because of the frequency with which mixed
infections are found in birds reared in confinement, a drug
such as ASE was used to provide a broad-spectrum type of
protection. Recent surveys on the prevalence of coccidiosis
in pheasants (Norton, 1981) indicate that these drugs have
not been exerting the degree of control required to maintain
a low mortality rate. This would be expected with their
continued usage over a long period of time. Braunius (1982)
emphasized that a direct relationship occurred between time
of usage and number of Eimggig oocysts found. Norton (1981)
proceeded to test the efficacy in pheasants of several drugs
previously developed for use with chickens as well as two
drugs traditionally used with the pheasant. These drugs
included arprinocid, clopidol/methyl benzoquate, clopidol,
robenidine hydrochloride, monensin, methyl benzoquate,
halofuginone, ASE and sulfaquinoxaline. The results showed
that three of the new drugs, halofuginone, clopidol and
arpinocid, are more effective in reducing mortality,
maintaining weight gain and reducing oocyst output than ASE

and sulfaquinoxaline. Halofuginone (3 ppm) was able to

effectively control E. phagiani and E. dgodgnalis while

30
clopidol (125 ppm) and arprinocid (65 ppm) were most
effective against E. colchici.

Baidsaielesx

The distribution and severity of coccidiosis under both
field and pen conditions are dependent on oocyst survival in
the external environment and a host's capacity to protect
itself. Oocysts are able to sporulate and remain viable in
the presence of moisture, warm temperatures and good
oxygenation. In outdoor pens these conditions exist
primarily around watering troughs, in shaded areas such as
under trees (Farr and Wehr, 1949) and during the warm periods
of the year. Maintenance of well-drained, treeless fields
could reduce the number of infective oocysts in this type of
environment. Bejsovec (1973) noted a marked decrease in the
incidence of coccidiosis when pheasants were moved to large,
extensively drained, continuous field units. Changing of the
litter used in buildings keeps moisture and the number of
infective organisms at a desirable level. Regulation of
personnel movement within and between buildings reduces the
potential for introduction and spread of oocysts. Hosts that
are well-nurtured, free of pre-existing non-coccidial
infections and receiving an appropriate anti-coccidial drug
while exposed to a moderate number of oocysts are best able
to develop an immunity which will maintain the coccidia at
the infection level, resisting progression to the disease

state. This is critical to the survival of the host since

31
those reared under conditions of strict sanitation have
little or no resistance (Horton-Smith, 1954). Introduction
of a non-immune host to out-of—door units may well result in
acute coccidiosis (Horton-Smith, 1947).

Gallinaceous birds such as chickens, turkeys and most
particularly pheasants are very sensitive to alterations in
their environment. Movement of a flock, spexing, crowding,
abrupt change in lighting conditions and exposure to hard
rains may serve as environmental stressors producing a "fear-
like" response called the General Adaptation Syndrome (GAS)
(Hill, 1983). The physiological response in humans to
physical stress is a marked increase in the secretion of
corticosteroids, most particularly cortisone, initiated by
nerve impulses in the hypothalmus. A similar response in
birds to stress, i.e. elevated serum cortisone levels, has
been substantiated by researchers such as Monjan (1977). The
consequence of increased cortisone levels in both humans and
birds is suppression of the immune response. In humans this
temporary loss is sometimes followed by fulminating spread of
infectious disease in the body (Guyton, 1963) and could
potentially be the same in birds.

The coccidia appear to be most susceptible to climatic
conditions that reduce available moisture. Farr and Wehr
(1949) showed that oocysts on bare soil exposed to direct
sunlight did not survive more than 25-30 weeks, while those

with some protection from the drying effects of the sun

32
remained infective nearly twice as long. Exposure to
temperature extremes when moisture content is high has little
adverse effect on their viability. Bejsovec (1973) reported
that in Czechoslovakian range pens the highest incidence of
pheasant coccidiosis occurred during the winter months.
Chang (1937) found that unsporulated oocysts of E. mug
could survive in a water bath of 46°C for 245 minutes while
Pellerdy (1965) found that E. teggllg oocysts were able to
survive no longer than 60 minutes at 25°C in an incubator.
Survival of the coccidial oocysts is at a peak during those
times of the year when the environmental moisture content is

high, such as during rainy periods.

MATERIALS AND NETEODS

n ns de 'ons

The pheasant-rearing facilities at Dansville include two
buildings for indoor confinement and several wired padlocks
for outdoor maintenance. Each building is partitioned into
six brooder units, three of which will house a single hatch
during its 12—week indoor period. These rooms, labeled A-C,
are of increasing size to allow for growth of the birds
(Table 2). Room temperature is initially maintained at 95°F
then decreased weekly by 5°F until week 5 or 6 after which no
heat is added. The floor of each unit is covered with a 6~
inch layer of wood chips. Areas of the litter that become
wet are removed but otherwise no change is made until the
hatch has been moved to the next unit. At that time the unit
is emptied of equipment and litter, washed thoroughly,
disinfected with an ortho-phenol (Chematrol) or a quaternary
ammonium compound (Microphene) then allowed to remain empty
for one week.

Pheasants are moved at 12 weeks to initial outdoor pens
containing water troughs and feeder units of roughly the same
number as found indoors. These pens were created for 1-2
week adjustment to the new environment. The birds are then
moved to range pens which are larger and contain fewer water

33

34
troughs and feeder units. The pheasants remain in these

paddocks until released at age 18-22 weeks.

Table 2

'm ns ons of ndo r n' 8 nd ens

 

 

Dimensions (ft.)

 

 

 

 

 

Unit .Maia_822mll .Ben
A 40 x 50 4 x 6
a 40 x 150 8 x 10
c 40 x 200 10 x 12
x nt ns

For experimental purposes, a special pen was built
inside each of the three brooder units of one building.
Dimensions of the pens were chosen in such a way that the
same stocking density as in the main area was maintained.
Litter from the main area of a unit was added 2-3 times a
week to that in the special pen in order to ensure common
exposure to any parasites or other microorganisms that might

be present in the main flock.

d tc s
A single hatch totalled approximately 6,000 birds.
Twenty-six hatches were reared in a period beginning the
middle of March, 1981 and continuing until the middle of
December, 1981. Three separate hatches, one from each season

the 'put and take" program was in operation, were selected

35
for this study including hatch 2 (March 26-July 16), hatch 10
(May 21-August 27) and hatch 22 (August l3-December 4). Each
hatch was separated into two groups. The first consisted of
80 randomly selected, left-wing banded birds that were
isolated in the special pen (study group). This number was
maintained throughout the 18-22 week period by replacement of
dead or removed pheasants with birds randomly selected from
the main body of the hatch and banded on the right wing. The
remainder of the hatch (control group) was housed in the main
area of a unit. All birds in both groups were fed a mash
containing 28% protein, 7% crude fiber and 2% crude fat. The
coccidiostat Amprolium+ (see Appendix) was added to the feed
of the pheasants in the control group at a concentration of
125 ppm for the first 8 weeks and 40 ppm thereafter until one
week before their release at which time the drug was
withdrawn. This was done in keeping with the standard
management program at the farm. No coccidiostat was added to
the feed of birds in the study group. In all other ways the
two groups were treated similarly. Every week four birds
from each group were randomly selected, killed and
individually weighed. A Mettler balance was used for birds
up through 6 weeks of age, while birds 7 weeks and older were
weighed in a Chatillon scale. Necropsy was performed using
the method described by Stubbs (1954). Once the intestines
were removed from the body cavity, they were examined for

gross lesions; representative pieces of tissue were then

36
selected for subsequent study. One section from each of five
areas of the gut (distal half of the duodenum, jejunum, upper
ileum, caeca and lower ileum) was fixed in 10% neutral-
buffered formalin for histopathological examination while a
second section was taken for parasitological examination of
its contents. Feces from all four birds were pooled for an

oocyst count.

fiistopgthglogy

The formalin-fixed specimens were treated with alcohol-
xylene for 12 hours then embedded in paraffin blocks, each of
which contained a sample of the same gut area from all four
birds in a group. Tissue sections of 6 um in thickness were
fixed to a slide then stained with Ehrlich's hematoxylin and
eosin.

Microscopic observation with the 10x objective was used
to assess the condition of the tissue, note possible invasive
cells and detect gamonts and oocysts. Detection of smaller
parasites, differentiation of developmental stages and
determination of the parasite's position within the cell was

done with the 40X objective.

Eaggsigglggy

The intestinal contents of each area of the gut from the
four birds in a group were pooled. Microscopic examination
of the contents was made on direct smears and following sugar

flotation. Dimensions of a minimum of 10 oocysts for each

37
species observed were measured using an eyepiece scale that
had been calibrated with a stage micrometer. Other oocyst
characteristics such as shape, presence or absence of a
micropyle, polar granules and cell wall features were noted
for species identification.

Sporulation studies utilized a pool of all intestinal
contents from the four birds within a group. The material
was washed three times with tap water then placed in a petri
dish. Potassium dichromate (2.5%) was added to a depth of
one-quarter inch. At specified time intervals (24, 48 and 72
hours) a sample of the material was transferred to a centri-
fuge tube for sugar flotation and subsequent microscopic
examination. The percentage of sporulated oocysts for each
species was determined by counting a total of 100 oocysts or
by counting the total number on the slide if fewer than 100
oocysts were present. Sporocyst characteristics, e.g. stieda
body, granules, shape and size, were noted at this time.

Oocyst counts were performed on feces from the following
pheasants: study group: four live birds selected weekly and
all dead birds; control group: four live birds selected
weekly, any dead birds requested for special studies and any
live birds requested for special studies. An adaptation of
the McMaster's method (Technical Bulletin, 1971) was
utilized. Saturated zinc sulfate (33%) was added to the
feces in a ratio of 14:1. The well-mixed sample was filtered

through a sieve covered with a double layer of cheese cloth.

38
A McMaster's counting chamber was filled with the filtered
sample then allowed to stand for 5-10 minutes while the
oocysts rose to the surface. The total number of oocysts in
both sides of the chamber was multiplied by a factor of 50 in
order to obtain the number of oocysts per gram of feces (see

Appendix).

Buidemielesx

Dansville farm personnel maintained a daily record of
unit conditions and number of mortalities while the pheasants
were confined indoors. Those conditions noted included room
and floor temperatures, brooder stove settings, variable fan
settings, type of feed and outside temperature and weather
conditions, e.g. cloudy, humid, hard rains. Causes of death
were recorded when due to accident, starvation, drowning or
an animal kill. Those deaths that appeared to be disease—
related were diagnosed by DNR veterinarians following
necropsy of a representative few dead pheasants. Mortality
rates were based on the total number of birds originally
present within a group. Records during outdoor confinement
included type of feed and the number of deaths with comments
regarding cause if known.

Records of daily high and low temperatures and rainfall
for the months of March, 1981 through December, 1981 were
obtained from the National Weather Bureau located at the

Lansing Airport, Lansing, Michigan.

RESULTS

Qgccidial spgcigg
Four species of coccidia were identified: Eimggig
d__e_e__l_i_u 6 na 8. E. mines. E. abaaiani and E. salable};

Identification was based on oocyst dimensions (Table 3),
oocyst morphology (Table l) and location within the intestine
when possible. The size of the oocysts fell within the

reported values for each of the species.

 

 

 

 

Table 3
MW
Obemwed
lkmsunmmmts qunted
W1). W
(Inchlku. lxmgth tfidth Elape Ixmgth tfidth taupe
M W
E.duodenalis 22.6 20.0 1.13 20.3 18.1 1.10 Norton,
1967a
E.phasiani 25.3 18.1 1.40 24.7 17.1 1.45 Trigg,
196k:
E.pacifica 23.7 19.4 1.22 21.1 17.5 1.20 Omsbee,
1939
s. colchici 29.8 18.6 1.60 28.7 17.3 1.64 Norton,
1967b
C S u ts

The number of oocysts per gram of feces was determined
for both groups of batches 10 and 22 beginning at week 7 when
the birds were large enough to provide sufficient fecal

39

40
material. An oocyst count was also done on fecal material
from any dead birds within the study group that were received
from the Dansville farm. Eflevated oocyst counts paralleled
movement of the pheasants to the C unit and to the North-D
pen (see Figure 3). This was true with both the study and
control groups although the increase in the oocyst count was
not as conspicuous in the latter group. The same correlation
could not be seen with morphology except when the birds were
in the outdoor unit. Most of the increased or high oocyst
counts indicated a severe infection which was substantiated
by evaluation of tissue sections. Oocyst counts from birds
in hatch 22 were usually greater in number and increased more

frequently than in hatch 10.

ne a 0 an

Live pheasants delivered to the Animal Health Diagnostic
Laboratory from the Dansville facilities generally arrived in
satisfactory condition. They appeared alert with good
feather growth and no lesions other than those caused by
injury or cannabalism. On two separate occasions live birds
were selected from the control group for special studies.
Both times the birds were lethargic, small in size and had
diarrhea. Most cases of coccidiosis in this investigation
though were subclinical, diagnosed only following necropsy
and examination of the tissues.

Each bird was weighed following euthanization and the

weekly mean weight per group calculated. No weight

41

Figure 3: % Mortality and Oocyst Count for Each Week

of a Hatch, Both Groups

42

-—-Oocysi Count

WW \- LL
W’TYNjTIII'IIITI‘\TIIT“TIIIIIIITIIIII\‘IIY\‘UIIVVIUFI

/
\

l7

 

CONTROL GROUP

 

HATCH ‘2
HATCH ‘Io
HATCH‘22

  

n 1213511553

IO

Age of Birds (weeks)

23456789

1
I

Lll‘\111\\jlllLlllllLllllellLLLLllllllllll llltellll‘lllllllLfl
\\ it \Y V1 \‘ \‘

(°/.) Munuow —

---Oocysl Count (oocysts/gm feces)

 

 

\g ‘L A ‘\
tithirvaIirrrrlwti 1IIY‘\III\‘IIIIIIIII

  

EIBTIIEBMEEW

Age of Birds (weeks)

/
\
O.
D
O
(I
O ‘
>_ 4ND
s
I‘— {No
(D O N
N — N on
c O I
I I I l"
E i3 f3 n
4 < 4
I I I Do:
I lllLlllL-llllllllJLj Lil‘dlllfi llJlJJAIIL JillklllkxllllllliJ
\ V‘ “ \‘ \\ \\ \\

 

 

(°/.) Kulouow —

43
differentiation by sex was made. A frank decrease in the
mean weight was seen between weeks 11 and 14 for both groups
in all hatches (Figure 4). Within the study groups of
hatches 10 and 22, the weight loss corresponded with an
increase in mortality and in the number of oocysts present in
the feces. A decrease in weight gain and an increase in
mortality were observed in all hatches, both groups, with the
exception of the study group hatch 22, when the birds were

moved into the C unit and the North-D pen.

ss ' ns

No lesions of the mouth were seen nor were any of the
crops packed with wood chips. Macroscopic lesions in the
intestines and caeca consisted of enlargement of the blood
vessels, thickening of the intestinal wall, mucous covering
on the outer surface, watery intestinal contents and/or
hemorrhage and a white pasty material in the caeca. The
lesions were usually mild in degree and were seen infre-
quently in hatches 2 and 10 while appearing consistently
following week 3 in hatch 22. An increase in the intensity
of the lesions, i.e. involvement of more areas of the gut,
rather than a change in type, accompanied the few severe
infections. However, birds that died at the Dansville farm
often showed areas of the upper intestines in which the
intestinal wall was very thin and heavily covered with mucus.
These areas appeared black in color due to a granular

material resembling coarse sand which was visible through the

44

Figure 4: Mean Weight of Birds (grams) as Measured per

Week of Hatch.

Solid Line = Control Group

Broken Line = Study Group

(grams)

Meon Weight of Birds

700

400

I00
ISOO

q
0
O

A
O
O

IOO
I300

IOOO

700

400

I

 

4S

HATCH “'2

 

HATCH “Io

 

HATCH ‘22

 

1 1 I 1 1 1 A 1 1 \

56789ioui2I3i415I8’

Age of Birds (weeks)

to
03-:
p—

46
thin wall. Similar involvement of the caeca occurred on
occasion. In the rectum, a thick, white, occasionally blood—

tinged material was present.

Wingless

Microscopic examination of the tissues showed a mild
increase of red blood cells within the capillaries,
infiltration of granulocytes and slight damage to individual
epithelial cells in light coccidial infections of 10 or less
parasitic forms per field with 10x objective (low power).
Moderate infections of 10-30 parasitic forms per lpf (low
power field) included a moderate increase in red blood cells
between the villi, occasional red blood cells within the
tissue and a marked increase in mononuclear and granulocytic
leukocytes. Eosinophils were prominent in E. W1
infections, heterophiles in moderate infections with all
species and lymphocytes in infections with more mature forms
(gamonts and oocysts) of all species. Heavy infections with
schizonts, gamonts and oocysts (greater than 40 parasitic
forms per lpf) produced a marked increase in all blood cells
with some loss of villi architecture and shredding of villi
tips. Clubbing of the villi was occasionally noted. Some
segments of the intestinal tissue from birds that had died of
coccidiosis at the Dansville farm were fragile, necrotic and
contained no observable parasitic forms. Fibrosis was
observed occasionally in younger birds but more frequently in

those 8 weeks and older.

47

Eimgria phggiggi was identified in infections involving
the whole tip of a villus (Plate 1a) or those in which the
epithelial cells lining the villus were infected all the way
to the base (Plate lb). All forms of the coccidium including
schizonts, gametocytes and oocysts were present. Infiltra-
tion of the tissues with eosinophils aided in the
confirmation. In fact, the presence of this granulocyte, in
the absence of coccidial forms, was considered suggestive
that E. 911m had recently infected a bird or would be
found in another section of the gut. A finding of the
oocysts in the intestinal contents and/or the presence of
parasitic forms in another area of the gut substantiated this
conclusion.

EimgLia gglchici was identified with certainty when
schizonts were present in the caecal glands and cautiously
when in the glands of the jejunum and ileum. In heavy
infections, E. W can potentially invade this deep.
However, schizonts were observed infrequently in the glands
of the small intestines and the numbers were few. Concomi-
tant infections of the caecal glands (Plates 1c and 1d) led
us to conclude that they were the schizonts of Eh gglghigi.
The white pasty material occasionally found in the caeca was
considered almost diagnostic of an ongoing infection or one
near its end.

Coccidial forms confined to the epithelial cells lining

the proximal or upper portion of the villi could have been

Plate 1a:

Plate lb:

48

Photomicrograph of a transverse section of the

upper ileum showing Eimggig phggiggi in various
stages of development within the tip of a villus.

10X

Photomicrograph of a transverse section of the

jejunum showing Eimggig phggiagi lining the sides

of a villus to the base, various stages. 40x

49

 

Plate 1b

50

Plate 1c: Photomicrograph of a transverse section of the

caecum showing m MEI. in the glands. 10x

Plate 1d: Photomicrograph of a transverse section of the

caecum showing Emu W in a gland. 40x

51

 

Plate 1c

 

Plate ld

52
often in mixed infections so that identification of each had
to be made from the sporulated oocysts. Week 6 of hatch 2
was the only time that E. duodenalis was observed by itself.
All coccidial forms were present in a position above the
nucleus of the epithelial cells lining the upper half of the
villi in the duodenum and the jejunum. It was concluded that

these could very well be stages of E, duodgnglis.

'de 'o

Records of each coccidial species observed on a weekly
basis show that E. duodenalis was present in both the study
and control groups of each hatch for the same number of weeks
(4, 5 and 3 weeks, respectively). The infections appeared no
earlier than 5 weeks and were seen sporadically over the 18
weeks each hatch was studied. EMA dugdgnalis was the
first species observed, which was in late April. Eimggia
pgggiggi was seen most frequently. It affected birds of all
ages, both groups, beginning at week 7 of batch 2 and week 3
of hatches 10 and 22. It appeared with somewhat greater
frequency in the study group than the control group. Another
species frequently seen was E. m. It appeared in
hatch 2 at week 8 for both groups but in the ensuing hatches
was present by week 3. It affected birds of all ages. On a
'weekly basis it was seen less often than E. pnggiagi, most
notably in hatch 2, and somewhat less frequently in the
control group than the study group. Elmegia cglchici, the

most pathogenic species for young pheasants, was seen rarely

53
in the control group of all hatches (0, 2 and 2 weeks,
respectively) and seldom in the study group (1, 5 and 4
weeks, respectively). It was not until hatch 22 that younger
birds were affected and then only at week 4, both groups,
following movement into the 3B unit. Infections with this
species were confined to birds housed indoors. It was not
observed in either group previous to movement nor following
movement into the North-D pen.-

During the week previous to movement of the pheasants
into the outdoors frequency and intensity of coccidial infec-
tions was usually low. Eimggig phagiggi was in all infections
and E. pacifica was observed once. The week of movement, E.
phggiggi infected the study group birds of all three hatches
and the control group birds of hatches 2 and 22. Eimgrja
pgcificg was not seen at all in hatch 10 but was present in
the study group of hatch 2 and both groups of hatch 22. One
week following transfer to the outdoor pen, both species were
observed in all hatches, both groups and Eimggig ngdggalis
was present in both groups of hatches 10 and 22 as well.

Overall weight gain was greater in the control group of
hatches 2 and 22 than in the study groups of the same
hatches. In hatch 10, however, the weight gain for birds in
the control group was lower. Individual weight gain losses
and frank losses on a week-to-week basis tended to be
associated with movement of older birds into a new environ-

ment (unit C and the North-D pen) and/or multiple infections,

54

usually of three species or more. An actual weight loss
occurred in the control groups of hatches 2 and 10 and in the
study group of hatch 22 at the time they were moved to the
outdoors. The infections in the control birds were mild but
those in the study group of hatch 22 were more severe and
mixed.

Mortality records for control group birds indicate cause
of death only when many died in the same period, e.g. 400
deaths in hatch 10 during weeks 1-3 due to wood-chip blockage
and 95 deaths in hatch 22 during weeks 11 and 12 due to
starvation. Cause of death while in the North-D pen was not
reported, however, constant rains were recorded for each
hatch during this period. The number of deaths were as
follows: hatch 2 (128), hatch 10 (106) and hatch 22 (1428).
Outdoor temperatures at the Dansville farm were not monitored
but climatological data from the National Weather Service at
the Lansing, Michigan Airport shows average °F temperatures
of 69, 72 and 50, respectively, for the same periods.

Maximum and minimum temperatures are shown in Table 4.

 

 

 

Table 4
tu s u n m n
W
Hatch Maximum Ayggggg nigimum
2 79.8 69.2 57.8
10 83.2 71.9 60.2

22' 60.2 49.5 38.6

 

55
Cause of mortality within the study group was determined
by examination of all birds that had died at the farm. Table
5 shows the percentage of total deaths due to coccidiosis in
birds 8 weeks or less and the percentage of the original 80
birds that died from coccidiosis. The remaining deaths were

due to wood-chip blockage, starvation or bacterial infection.

 

 

Table 5
u nc cc'd n s n ds
W
Batch 2 Eatgh 19 Hagen 22
t.o tn 0

Total I: of Birds 64 64 60 60 64 64
Exmmhmd
Total 4 of Birds 33 24 41 33 38 25
with Parasite in
Thane
% of Total 52 38 68 55 59 39
t of Birds 8 Weeks 12 ll 21 19 23 15
orlfissvfith

Paanfiteinwrumme

% of Tbtal 19 17 35 32 36 23

 

DISCUSSION

99cc1dial Epgcigs

Identification of the four Em species was based
primarily on oocyst dimensions and morphology. Usually one
or more features of the sporulated oocysts were quite
distinctive and the species thus readily identified. Similar-
ities between two species in shape, size or cell wall
characteristics necessitated the use of other criteria such
as location within the gut, tissue and/or epithelial cell.

Microscopic examination of unsporulated oocysts made
differentiation of E. duodgpglis and E. pacifica difficult
unless the material in which the oocysts were found was taken
from the caeca. E. dggdgngns has not been reported in this
area of the gut (Norton, 1967a) whereas E. M has
(Ormsbee, 1939). Observation of sporulated oocysts revealed
features distinctive for each species. The wall of E.
pgcifica was thicker than that of E. dggdgnalig and on
occasion was striated. This feature was first reported by
Wacha (1973) having been missed by Ormsbee (1939). Wacha
found that these striations were not consistently present on
all E. pgciflca oocysts which would explain findings of only
a few striated oocyst walls. A light thinning of the inner
wall near one end of the E. 235112.123 oocysts gave the

56

57

impression that a micropyle or operculum was present although
in fact this species has neither. The wall of E. dgggegglig
oocysts was of an even thickness throughout. Although the
sporocysts of both species has a “stieda" body that of the E.
dugdgnglis sporocysts was very well defined. Wacha (1973)
reports that this species also has a 'substieda' body, which
may aid in its distinctive appearance. Differentiation of
these two species in any area of the gut other than the caeca
was not possible by this investigator.

Eimggig phggiggi and E. gglchici oocysts, although
similar in appearance, have characteristics individualistic
enough to allow for differentiation. Our observations showed
that both were elliptical in shape, but that E. gglghigi was
usually longer and narrower. Its cell wall was thinner and
the flattening on one side, an oocyst feature of both
species, was more distinct. The sporocysts of E. W
appeared more tapered even though both species have a well-
defined "stieda" body.

Differentiation within the tissue was based on the
general rule that E. £21m is a glandular parasite
(Norton, 1967b) whereas E. pnggiagi is confined more to the
epithelial cells covering the villi, often the tips (Trigg,
1967a). Two exceptions to this rule tend to complicate
separate identification of the two species by an
inexperienced investigator: (l) the large first-generation

schizonts of E. phggiggi locate in the glands of the upper

58
half of the small intestine where first and second generation
schizonts of E. cglchici may also be found and (2) the
gametocytes and oocysts of E. gglgnigi are found in the
epithelial cells and lamina propria of the caecal villi where
they may be mistaken for E. phgsigni. Schizonts and
gametocytes in both the lamina propria and epithelial cells
lining the villi of portions of the gut other than the caeca
were considered confirmation of an infection with E.
pnggiggi. No stage of E. duodggglig invades the lamina
propria and schizonts of E. cglchici are found deeper in the
tissue. All stages of E. 2391M have been reported
(Ormsbee, 1939) to infect only the epithelial cells lining
the villi throughout the small intestine and proximal half of
the caeca. Identification of E. colohici was substantiated
by the presence of schizonts in the caecal glands on six
separate occasions: hatch 2 (week 11), hatch 10 (weeks 6, 7,
8 and 9) and hatch 22 (weeks 4 and 11). The oocysts were
present in the intestinal contents without observable tissue
involvement three times: hatch 10 (week 10) and hatch 22
(week 9 and 13). It was inferred that at these times the
prepatent period had been concluded. The few infections seen

were primarily in the study group (10:4).

W
The infections seen for the most part were subclinical
and mild. Damage to the intestinal tract occurred, but only

on occasion were the resultant lesions life-threatening. The

59

numbers of parasites within the tissues were generally higher
among young birds who tend to be more susceptible to
infection than immune older birds (Long and Horton-Smith,
1968). Mortality among this age group, however, was
relatively low except at week 2, an age when the birds are
apt to incur a wood-chip blockage and/or become infected with
E. cglcpici. This organism was not observed in the intes-
tinal contents or tissues of the 2-week old birds we
examined. None of the chicks appeared lethargic nor did they
fail to gain weight. This does not rule out though the
possibility that E. gglghigi was a cause of death since none
of the dead birds at this age were made available to us for
examination. It had been assumed they had died of wood-chip
blockage.

An increase in mortality often occurred following
spexing and movement into the C unit (week 7) and movement
into the North-D pen (weeks 12 and 13). Usually an increase
in the oocyst count paralleled the increased mortality
suggesting that the deaths were related to coccidiosis, which
was confirmed by parasitological and histopathological
studies. Movement is stressful to the birds and may
stimulate physiological changes within the pheasant which
allow a pre-existing infection to intensify (see below).

All birds within the control group were receiving the
anti-coccidial drug amprolium+. Trials have shown that

amprolium is effective in the control of E. o c c'

60

coccidiosis (Norton, 1967b). Amprolium+, because of the
synergistic effect of attentuating ethopabate with amprolium,
could have a mode of action and efficacy different than that
of amprolium alone. The drug did appear to be effective,
though, against E. gglchici a pathogen of 2-4 week old
chicks. This organism was able to cause death in 100% of
unmedicated birds with a dose of 80,000 oocysts (Norton,
1981) yet it was implicated in very few of the infections
found in our control group birds and only once in chicks 2-4
weeks old (hatch 22, week 4). This may be an indication that
the coccidiostat was able to prevent the parasite from
developing to the oocyst stage thus reducing the number of
oocysts in the environment and affording little opportunity
for reinfection. A moderate number of infections caused by
E. cglchici were observed among our study group birds of
batches 10 and 22 while housed primarily in unit C. Only
once was the parasite seen in hatch 2 which was one week
previous to being moved outdoors from the same unit. It
could be inferred that the parasite had been introduced into
the building from an outside source while the pheasants of
hatch 2 were housed in the C unit or that the coccidia had
survived the winter. Although infections were often severe
among the non-medicated birds, none were lethal indicating a
good immunity had been established in these older birds.

Bimsria ahaaiaai. a pathogen of young pheasants (Trigg.
1967b), was observed frequently throughout these studies.

61

Its first appearance in hatch 2 occurred while the birds were
housed in the C unit but in hatches 10 and 22 it was first
observed while the pheasants were in the A unit. The earlier
appearance of the parasite in the later hatches may relate
once again to management procedures. If the C unit was not
disinfected as carefully as those units housing the younger
pheasants, then oocysts may have survived the winter.
Introduction of oocysts into units A and B by workers, once
the rearing had begun, would then account for the appearance
of E. phagiggi, and possibly other species in young chicks of
the summer and fall hatches. The appearance of E. ghagiggi
in pheasants of all ages is indicative of the mediocre
protection afforded by the coccidiostat against this species.
Since the mortality rate among both groups was relatively low
while the pheasants were housed in the A and B units, it
seems likely that the coccidiostat played no more than a
minor role in controlling the disease. A low to moderate
number of available oocysts was probably a more important
factor (Long, 1973) a condition that could be related to
immunity as the birds aged.

No drug trials with E. 9.8911122 have been reported in
the literature. This species was also seen frequently which
would suggest that amprolium+ may not be the drug of choice.
Infections in which E. dggggnglis was one of the causative
agents were few to moderate in number. They occurred in

study group birds and control group birds with nearly the

62
same frequency, which once again suggests better control
through immunity than the coccidiostat.

Several investigators have studied the effect of host
age on the reproductive and pathogenic potential of Eimggig
species (Rose, 1973; Krassner, 1961; Tyzzer, 1929; and
Horton-Smith, 1947). Conflicting results indicated greater
susceptibility in both younger and older birds. Long and
Horton-Smith (1968) provided some clarification of the
problem by suggesting that susceptibility to clinical disease
was greater in young birds while susceptibility to oocyst
reproduction was greater in older birds. Our data indicates
greater number of developing coccidia in the tissue, lower
mortality and less weight-gain reduction in birds 2-6 weeks
of age. The number of infected birds increased between
hatches 2 and 10 which suggests that the number of oocysts
within the unit was increasing. This could be due to
contamination from another source, increased number of shed
oocysts and/or conditions within the unit more favorable to
oocyst development and survival such as increased humidity
and temperature. The number of parasites seen in these young
pheasants, although many, was still not sufficient to cause
clinical coccidiosis. Environmental conditions within the A
and B units where birds of this age were housed were perhaps
less stressful or birds of this age may not be as susceptible
to the effects of stress. The less severe infections seen in

the older birds could have been related to oocyst dose sizes

63
that were smaller on a relative basis (# of oocysts/4 of host
tissue cells) and an immunity that should have been
established by this age. Movement of the older birds
appeared to be the most critical factor in the development of
disease as judged by weight-gain reduction, weight loss,
elevated oocyst counts and increased mortality during these
periods. Stress-related immuno-suppression would account for
the dramatic increase in susceptibility among these otherwise

resistant hosts.

W

W 211.811.1111 was observed in all coccidial
infections during movement and confinement of the pheasants
to the North-D pen. Eimgzia pagifiigg was seen in 50% of the
groups within 2-3 days and, by the second week, E. dggdgnalig
was present in all groups except those of the spring hatch.
The pathogenicity of E. pagifiigg is unknown, but its
potential as a contributor to the coccidial-related deaths
that occurred with increasing frequency from spring to fall
is suggested by its appearance in many of the diseased birds.
Its action may be an enhancement of biological alterations
known to occur within the host as a result of stress-induced
coccidiosis or it may help to impair development of immunity,
a phenomenon of mixed coccidial infections suggested by
Rommel (1970c). There is little doubt, though, that E.
M is the species primarily responsible for the high

mortality rate during periods of hard rains among pheasants

64
confined to the North-D pen. Reported to be a pathogen of
younger pheasants (Trigg, 1967b), E. phagiggi would probably
inflict only a moderate degree of damage were the birds not
severely stressed.

Hill (1983) writes that physical stress can effect
within an animal a chain reaction of biochemical events
terminating in suppression of the immune system and an
altered glycemic state. The initial stimulus for these
complex changes is increased activity of the sympathetic
nervous system which affects many functions of the body.
Three of these have been related to the General Adaptation
Syndrome, which is a group of different stress-related
physiological reactions: (1) stimulation of the adrenal
medulla with resultant increase in nemepinephrine and
epinephrine (Zachariasen and Newcomer, 1975), (2) stimulated
release of pancreatic glucagon (Freeman and Manning, 1976)
and (3) release of adrenocorticotrophic hormone which acts on
the adrenal cortex to increase production and secretion of
corticosterone. The key role of corticosterone in stress is
suppression of the immune system by its depressive effect on
the number of circulating leukocytes and on antibody form-
ation. As a glucocorticosteroid, corticosterone also acts to
a very minor degree with the other two stimulated hormones to
ultimately increase blood glucose concentration.

Hyperglycemia has been reported to be a consequence of

infection by Pratt (1940) and Herrick (1950), but in severely

65

diseased birds some investigators have found the reverse. In
their study on the physiological basis of mortality among
Egmggig ggnellg infected chickens, Witlock et al. (1981)
reported that hypoglycemia was one of the diagnostic features
of incipient death. Freeman and Manning (1976) found that
birds became hypoglycemic 30 minutes after they were stressed
by handling.

A consequence of hypoglycemia is hypothermia, glucose
metabolism being essential for maintenance of body
temperature (Freeman, 19718). Herrick (1950) reported that
coccidia-infected birds subjected to a cold environment
experienced a decreased metabolic rate and a drop of 20°C in
body temperature. Neither Keener (1963) nor Witlock et a1.
(1981) were able to substantiate these findings; however,
they did find that hypothermia was another clinical sign of
impending death among infected birds. The condition of
hypothermia in birds dying from coccidiosis may be
physiologically-based then rather than environmentally
induced.

The sequence of events leading to the high death rate
among those pheasants confined to the NorthéD pen may
theoretically occur in a somewhat complex manner. As a
consequence of the high number of oocysts washed down into
the pen, pheasants newly introduced to this area would in a
few days ingest high doses of them. The infection level

would increase both in numbers and intensity. Coccidia

66
utilize considerable quantities of carbohydrate during their
metabolism (Ryley, 1973) thus a transient reduction in blood
glucose concentration may be experienced by these hosts.
Hypoglycemia is another pathway for stimulation of the
sympathetic nervous system (Guyton, 1963). In this manner,
glucose is released from glycogen stores and metabolized from
other sources for the energy required during repair of tissue
and body temperature regulation. Hyperglycemia would ensue.
If at this point the animals were stressed by hard rains,
increased corticosterone would suppress the immune system.
The coccidia would then be free to reproduce unhampered,
overwhelmingly the activity of the medication and inflicting
irreparable damage to the intestinal mucosa as was seen in
birds that died at the farm following hard rains. Proteins,
fluids, nutrients and blood would be lost. Glucose would be
utilized rapidly to meet energy requirements of the host
*while little or none could be absorbed from the intestines
leading to hypoglycemia and eventual hypothermia. Both of
these conditions, as well as the diminished renal function
noted by Witlock et al. in dying birds, are signs of shock
resulting from reduced cardiac output (Guyton, 1963). It may
be concluded then that the pheasants are dying from irrevers-
ible shock enduced by the sequence of events following the
stress of exposure to hard rains. Environmental tempera-
tures, although not responsible for the initial hypothermia,

may, if low, intensify a state of reduced body temperature.

67

Guyton (1963) reports that during shock the body temperature

tends to decrease even more if the subject is exposed to even

the slightest cold. This would support Herrick's findings

and may explain, in part, the higher mortality rate among the

pheasants in the cooler months.

8.

SQEEQL!
Four species of coccidia were found to infect the
pheasantS: 5.1mm W: 21. 2111113111. E.-
aasifise. and E. 9.91211121-
Most infections were mixed but usually did not progress
to the disease state.
The infection level was more intense in younger birds
while mortality was higher in older birds.
Infection level and mortality were higher in the non-
medicated group.
As the seasons progressed from spring through fall,
infections were incurred at younger ages and by more of
the pheasants.
Mortality was highest in both groups following movement
into the C unit and the North-D pen.
Eimggig gglghigi, a coccidial parasite of pheasant
chicks 2-4 weeks old, was rarely seen in birds of this
age.
Eimggig phggiggi was seen most frequently and appeared
to be the species with the most serious effect on the

pheasants once transferred to the outdoors.

10.

11.

12.

68
m m was also observed frequently but
pathogenicity studies are required before its potential
as a cause of death among the pheasants can be truly
ascertained.

The coccidiostat amprolium+

appeared to be quite
effective against E. gglghjgi_but only mildly so against
the other three species.

Hard rains are stressful to the pheasants who tend to be
more heavily dosed with oocysts while in the North-D pen
thus loss of immunity is of greater consequence.

Cooler temperatures could increase the death rate among

birds stressed by hard rain.

RECOMMENDATIONS

Medicate the birds with a new drug that maintains the
efficacy against E. lechici while being effective
against E. phggiggi and E. pagifiga.

Reduce the number of oocysts in the initial range pens,
e.g. the North-D pen, either by the addition of a
drainage system or by treatment of the soil with a
chemical that is destructive to coccidial oocysts
without adversely affecting the pheasants.

Clear the field of all vegetation or other matter that
might provide the oocysts shelter from the sun. It is
understood that this cannot be entirely effective since
the buildings which are close to these pens shade the
area rather extensively.

Routinely increase the drug dosage for the two weeks

that the pheasants are in these pens.

69

APPENDIX

2.

APPENDIX

Additional information on management techniques at the

Dansville farm.

a. Any individual entering an inhabited indoor unit is
required to place disposable plastic boots over
his/her footwear then step into a liquid
disinfectant.

tn Previous to transfer of the pheasants into the
largest indoor unit (C), plastic blinders called
'spex' are placed on the bird's beak with a plastic
pin inserted through the nasal passage to hold the
"apex" in place. These restrict forward vision,
thereby reducing the tendency of feather-picking
and cannibalism. The 'spex' are removed just
before the birds are released. (Martin Pollok,
"Pheasant Rearing,” Michigan Department of Natural
Resources.)

c. Transfer of the pheasants from the largest indoor
unit into the first range pen is done at night so
that the pheasants can adjust gradually to the
change in light.

Amprolium+: Amprolium attenuated with ethopabate.

70

71
Modified McMaster's formula for calculating # of
oocysts/gm feces.
a. 2 gm feces + 28 ml saturated zinc sulfate=30ml
total volume of sample
b. 0.15 m./sq.cm in counting chamber
c. 2 sq.cm counted

6. formula

meagre—ts counted x MW .. t of oocystS/gm feces
(0.15ml/8q.cm)(289.cm) 29m feces

OR

4 of oocysts counted x 50 = t of oocysts/gm feces

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VITAE

The author was born in Ontanogon, Michigan on October 7,
1941. Her family moved to New York State when she was four
years of age and to Massachusetts when she was twelve. She
returned to Michigan at seventeen to attend Michigan State
University. Following graduation she served her internship
at E.W. Sparrow Hospital in Lansing, Michigan where she
remained employed as a Medical Technologist for one year. In
1973 she and her husband along with their two daughters moved
to Duluth, Minnesota. For seven years she worked in research
in the Departments of Physiology and Biochemistry at the
University of Minnesota, Duluth Medical School. In 1980 she
returned with her daughters to East Lansing, Michigan to
enter graduate school at Michigan State University where she
worked as a graduate assistant in the Medical Techology
Program while pursuing her studies and research. She is

presently employed by E.W. Sparrow Hospital.