OBSERVATIONS ON SOME FACTORS WHICH MAY INFLUENCE
USCEPTIBILITY OR RESISTANCE OF CHICKENS AND TURKEYS TO
HISTOMONIASIS

By
CLARENCE JOSEPH WELTER

A THESIS
Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health
19 59

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ACKNOWLEDGMENTS
The author wishes to express his sincere appreciation
to his major advisor, Dr, William D, Lindquist, for his
guidance and deliberative suggestions throughout this in­
vestigation, and for making available funds for the procure­
ment of experimental supplies, without which this work could
not have been done.
Gratitude is also extended to Dr. David T. Clark,
Department of Microbiology, for his generous assistance in
photographic work, to Dr. William D. Baten, Agricultural
Experiment Station Statistician, for his assistance in
statistical aspects of the problem, and to Dr. Jack J. Stock­
ton, Department of Microbiology, for his generous answers
to the author’s inquiries.
Finally, thanks are expressed to all those individuals
who have helped in any way toward the completion of this
investigation and the preparation of the manuscript.

ii

TABLE OF CONTENTS
PAGE

INTRODUCTION ...........................................

1

REVIEW OF L I T E R A T U R E .............

3

MATERIALS AND METHODS
A.

Experimental materials
1.
2.
3.
A.

3.
C.

D.

.................................
..........................

Chickens and t u r k e y s ........................
Feed
. . . . . . . . . . . . . . .
Infective materials
. . . . . . .
Other materials
. . . . . .
........... . .

1?
17
17
IB
19

Care, management, and distribution of chickens and
turkeys prior to the beginning of experiments

20

Infection of chickens and turkeys

20

. . . . . . .

1 * Per os i n o c u l a t i o n ..........................
2 . Rectal Inoculation ..........................

20
21

Exposure of birds to stresses

21

1.
2.

21

S t a r v a t i o n ...................................
Simultaneous Eimerla tenella infections in
c h i c k e n s ...................................
......................
3. Temperature variation
A. Anemia
5 . Fowl pox vaccination
.........
6 . Cortisone i n j e c t i o n s .................
E.
F.
G.
U.

17

22
22
23
23
23

Surgical transplant of cecal material
. . . . .
Serum processing and injection ..................
Post mortem examination
. . . . . . .
.........
Care of tissues for sectioning, and staining
methods

2A
25
26

R E S U L T S ................................................

28

A.
B.

27

Surgical Implantation of cecal material
. . . .
Effect of stress upon hlstomonlasis In chickens
and t u r k e y s ...............
Immunity and resistance
. . . . .
.............

3°
39

D I S C U S S I O N .............................................

>*7

S U M M A R Y ................................................

59

BIBLIOGRAPHY ...........................................

6^

A P P E N D I X ................................................
Hi

70

C.

28

LIST OF TABLES
TABLE
1*

PAGE
Histomoniasis in splenectomized chickens ana
in chickens and turkeys receiving surgical
implantations of cecal contents before in­
fection .................

2 . Histomoniasis
3.
4.
5.

in starved chickens and turkeys

3*
9.

10.

31

Concurrent infections of histomoniasis and
coccidiosis in chickens ...................

32

Hemoglobins of chickens and turkeys infected
.........
with Hlstomonas • • • • • • • • •

34

Histomoniasis

in anemic chickens and turkeys

6 . Histomoniasis
In chickens and turkeys exposed
to 4° C or 37° C t e m p e r a t u r e s .............
7.

29

35
36

Histomoniasis in fowl pox-vaccinated or corti­
sone-treated chickens and t u r k e y s .........

38

Histomoniasis in chickens and turkeys of
different a g e s ............................

40

Histomoniasis in chickens and turkeys receiv­
ing multiple infections of Hlstomonas or
serum from recovered b i r d s ...............

42

Histomoniasis in turkeys receiving immune or
non-immune chicken serum ...................

iv

46

LIST OF PLATES
PLATE

PAGE

I,

Lesions due to histomoniasis in a turkey . . .

71

II.

Heart of a turkey Infected with histomoniasis

72

III.

Proventriculus of a turkey infected with his­
tomoniasis

73

IV . Mesentery of a turkey Infected with histonionlasis
. . ............ . . . . . . . . . .

7^

V.

Lesions due to histomoniasis in a chicken

v

. .

75

INTRODUCTION
Histomoniasis (synonyms; blackhead, enterohepatitis)
was first described in turkeys by Cushman in 1893*

Since

that time the literature concerning this disease has in­
creased voluminously.

The majority of the investigations

have dealt with the etiological agent, mode of transmission,
pathology, treatment and prevention of histomoniasis in
turkeys.

Since the disease spreads rapidly and is highly

fatal to turkeys, most of the work has been done utilizing
this host.

Turkeys of all ages are susceptible, although

some workers consider the most dangerous period to be 4 to
25 weeks of age.

Mortality is quite variable, sometimes

reaching 100 percent.
The literature pertaining to fatal histomoniasis in
chickens has been scant*

The importance of this disease in

chickens has been minimized to the role they play in main­
taining and disseminating the etiological agent, and the
economic loss in this host is not often realized.

Although

fatal histomoniasis in chickens does occur, the benign form
is seen more often and is followed by recovery, even though
the lesions produced are quite similar to the lesions in
turkeys dying of the disease.

The ability of stress to

trigger fatal histomoniasis in chickens has been suggested,
1

2
although never adequately substantiated with controlled ex­
periments.

The mechanisms and circumstances responsible for

resistance of chickens and turkeys to histomoniasis and the
factors affecting this resistance have not been investigated.
The purpose of this investigation was to determine whether
various stress conditions (starvation, exposure to temper­
ature extremes, anemia, coccidia infection, vaccination,
cortisone) would alter the disease or mortality in chickens
and turkeys due to Hlstomonas.

In addition, an answer was

sought for the question of whether bacterial flora in the
ceca influenced growth of the histomonad or was In some way
responsible for the course of the disease.
immunity was also raised.

The question of

It had been shown by Sautter £t.

a l . (195°) that whole blood obtained from turkeys, recover­
ing from histomoniasis, would not protect susceptible
turkeys from contracting the disease.

In the present in­

vestigation an attempt was made to determine if serum, ob­
tained from recovered turkeys, Influenced the course of
histomoniasis in chickens, or if immune or non-immune chicken
sera protected turkeys from contracting blackhead.

An

attempt was also made to determine what effect age, previous
exposure, and splenectomy (in chickens only) would have
upon histomoniasis in chickens and turkeys.

REVIEW OF LITERATURE
Histomoniasis was first observed in turkeys by Cush­
man (1Q93)* and later the etiological agent was described
by Smith (1895) as Amoeba meleagrldls. a protozoan which he
observed could produce conditions in turkeys comparable to
amoebic dysentery in man,

Tyzzer (1920) noted the mastigo-

phoric and pleomorphic nature of this protozoan in the ceca
of turkeys and classified it as Histomonas meleagridls (order
Rhizomastigina).

Delaplane (1932) isolated flagellated cecal

protozoa, which produced fatal histomoniasis, when used to
infect other turkeys, thus supporting Tyzzer*s studies.

Be­

sides the flagellated stage, which possessed from 1 to k
flagella and occurred in the lumen of the turkey ceca,
Tyzzer (1919) observed aflagellated tissue forms which he
divided into three stages - invasive, vegetative, and re­
sistant.

The invasive stage was considered to be that

stage of Histomonas found In early lesions.

The cytoplasm,

containing inclusions, was basophilic and consisted of
clear ectoplasm and finely granular endoplasm.

The vege­

tative stage was larger than the invasive stage and appeared
in slightly older lesions.

The cytoplasm, without inclu­

sions, was still basophilic and transparent.

The resistant

stage, found in the oldest lesions of the disease, was
3

4
smaller than the two previous stages and possessed an acido­
philic cytoplasm filled with small granules or globules.
Other protozoa have been Incriminated from time to
time as the etiological agent of enterohepatitls.

Cole and

Hadley (1903) reported the coccldiura, Elmerla avium, as the
causative agent, and they maintained that the amoeba which
Smith observed was really a schizont stage of this parasite.
Similarities and dissimilarities between coccidiosis and
enterohepatitis were indicated by Schofield (1926).

Tri­

chomoniasis, another protozoan disease, has been confused
with histomoniasis.

Symptoms and pathology are somewhat

similar for both diseases.

Hadley and Amlson (1911) and

Jowett (1911) believed that blackhead was caused by trichomonads, but their work was repudiated by the works of
Tyzzer (1919* 1920).

More recently Allen (193^) has advo­

cated two types of enterohepatitis, one type produced by
Histomonas meleagrldls and the other by Pentatrlchomonas
galllnarum.

Lesions of both types may be differentiated

macroscopically (Santos 1 9 ^ » Allen 19^1)*

Other etio­

logical agents of histomoniasis besides protozoa have been
advocated.

Yeast-like fungi have been incriminated several

times in this capacity.

Enigk (1935) and Menzani (1939)

reported a blastomycete as the cause of the disease.

Kuprowski

(1955) regarded the hepatic lesions, characteristic of the
disease, as anemic infarcts.

He isolated Candida albicans

from the livers of two out of eleven turkeys dying from

5
blackhead, and considered this fungus to be responsible for
the disease.
The growth of Hlstomonas ly? vitro was first accom­
plished by Boeck (Tyzzer 193*0.

Using Locke-egg-serum

medium he Isolated Hlstomonas. together with bacterial flora,
from cecal discharges of a chicken.

Drbohlav (1924), also

using Locke-egg-serum medium, noted the importance of hydro­
gen ion concentration, as did Tyzzer (193^0 with his medium.
Bishop (1938) isolated and maintained Hlstomonas in Laidlaw’s
medium.

She observed, as did Tyzzer (193*0* that Hlstomonas

may be rendered non-pathogenic by propagation i n vitro over
a period of time.

Delappe (1953&), using Laidlaw’s medium,

demonstrated the use of antibiotics in facilitating in vitro
isolation of Hlstomonas.

He also observed the effects of

pH changes, age of original inoculum, anaerobic environments,
and oxidation-reduction potentials upon growth of the histomonad in culture (1953b* 1953°> 1953*1 )•

So ^ar no one

has been successful in growing Histomonas free of bacteria,
nor has culturing been very successful for diagnosis.
Histomoniasis is a disease of fowl not autochthonous
to any one part of the world, as affirmed by reports from
such widespread areas as Australia (Hungerford 1937)* Brazil
(Santos 1944); Dutch East Indies (Picard 1929); Germany
Enigk and Wetzel 1938)* Italy (Wenzani 1939); Japan (Nilmi
1930); Poland (Kuprowskl 195°) and Russia (Vorob'ev and
Kolotilov 195*+).

6
Most of the literature on histomoniasis has been con­
cerned with turkeys, mainly due to Its economic importance
in this host.
chickens.

However, it is also of primary importance in

It has been observed by many workers that chickens

experience a mild form of the disease, recover, and remain
carriers and disseminators of the disease.

Some investi­

gators noted that histomoniasis results in retarded develop­
ment of the chicken (Baker 1933) and sometimes mortality.
Kaupp (Eveleth 1943) observed the death of 42 out of 43
five-week-old Silver Spangled chickens.

Eveleth (1943) re­

ported an outbreak in chickens in which there was a loss of
250 out of a flock of 500 with more showing symptoms.
also noted retarded growth in infected chickens.

He

Kunger-

ford (1937) reported an epidemic in New South Wales which
killed 200 out of a flock of 640 chickens; in another flock
of 400, twenty-five percent died.

Eriksen (I92 5 ) stated

that losses may vary considerably in different flocks of
chickens.

Two flocks were observed to suffer losses of

more than 50 percent while other flocks, complicated with
coccidiosis or brooder pneumonia, also showed high mor­
talities.

It is the opinion of Lund (1956) that histomoniasis

in chickens is serious when their resistance is lowered by
other diseases, by vaccination, or by undue exposure to
adverse conditions.
Other hosts than turkeys and chickens have been
reported to be infected with histomoniasis.

Wenrlch (1943)

described the morphology of Histomonas from pheasants, and
Lucas £t. & jL.

(1956) reported the partridge as a host for

this organism.

In Russia geese were observed to be infected

(Vorob’ev and Kolotilov (195^)*

Other hosts are grouse,

quail, guinea hens, pea fowl, ducks and pigeons.
Transmission of histomoniasis may occur in several
ways, when considering both natural and artificial means.
Prior to the discovery by Graybill and Smith (1920) of the
role of the common cecal worm, Heterakls galllnae. in the
transmission of this disease, it was assumed that infection
by the consumption of freshly passed histomonads was the
common mode of transmission*

However, Graybill and Smith

(op . cl t. ) produced histomoniasis in healthy turkeys by
feeding them feces (containing embryonated Heterakls eggs)
of Infected birds.

Feeding bird feces, not containing

Heterakls eggs, failed to produce the disease in turkeys.
These workers, likewise, produced blackhead In turkeys by
feeding embryonated Heterakls eggs only, but they did not
determine whether the histomonad was carried along on the
outer surface of the egg shell or within the egg.
and Fabyan

Tyzzer

(1920) suspended embryonated Heterakls eggs in

1.5 percent nitric acid for 3 days to render them bacterlclogically sterile and, at the same time, destroy any his­
tomonads that might have been adhering to the outside of
the egg shells.

Exposure of the unprotected histomonad in

feces to 1.5 percent nitric acid was observed to be lethal

to the organism.

Upon feeding these "sterilized" embryonated

eggs to turkeys the disease was produced, therefore indicat­
ing the location

of the hlstononad.

The Ingestion of male

Heterakls or ncn-embryonated Heterakls eggs did not produce
histomoniasis (Tyzzer 193^)> nor did a ground up suspension
of embryonated eggs or larvae (Swales 19^8).

This indicated

that hatchable embryonated eggs were necessary for producing
the disease.

The histomonad itself has never been demon­

strated satisfactorily in the Heterakls egg.

Niimi

(1930)

claims to have seen the causative organism, but his work
has never been substantiated.

Many methods for demonstra­

tion have been tried by different workers, but to no avail.
Hlstomonas has been observed in the Heterakls larvae by
Tyzzer (19 3M, who pictured the histomonad in the intestinal
epithelium

of an eleven-day-old larva.

Swales (19^8) art­

ificially hatched embryonated eggs, sterilized the larvae
in 50 percent hydrogen peroxide, and introduced them directly
into the ceca of a turkey by means of a laparotomy to pro­
duce histomoniasis.

He concluded that the living larvae

must be present to Initiate the disease process, but upon
repeated examination of larvae was unable to demonstrate
the histomonad at any time.

Out of 100 worms from a turkey

dying of blackhead, Desowltz (195°) observed sections from
one showing 2 enlarged intestinal epithelial

cells filled

with amoebulae which he believed to be Hlstomonas.

Connell

(1950) observed in second stage Heterakls larvae abnormalities

9
such as cutlcular swellings and diffuse thickenings, and
suggested that they might be caused by a stage of Histomonas.
Although no stage of Hlstomonas has ever been satis­
factorily demonstrated in the Heterakls egg, it has been
observed by many workers that the organism, in this pro­
tected niche, is quite resistant to environmental conditions.
Graybill

(1921) stated that "infectious soil that had remained

unoccupied for a period of five months, beginning in the
depths of a severe winter, still harbored viable ova of
Heterakls and proved highly dangerous to young poults."
Four healthy turkeys which he placed in the
tion contracted histomoniasis.

yard

in ques­

Tyzzer (193^) > Van Es and

Olney (193^)* and Nilmi (1937) also observed this over­
wintering of the histomonad within the still viable Heterakls
eggs.

Farr (195^) seeded

outdoor plots of earth to a depth

of two inches with fresh droppings of chickens and turkeys
containing Hlstomonas and the eggs of several nematodes,
one of which was Heterakls.

After a period of 66 weeks

samples from these plots, when fed to healthy turkeys, pro­
duced histomoniasis.

During this period of time temper­

atures ranged from 5° F to 100° F.
Transmission of histomoniasis by the ingestion of
free histomonads in droppings probably occurs only rai'ely
The causative agent is unable to survive longer than a few
hours outside the avian host.

Tyzzer and Collier (1925)

stated that "the protozoan is discharged in a form that is

10
Incapable of surviving long outside the body, but which may
produce infection if immediately ingested."

Tyzzer (1932)

and others reported that very Irregular results follow
attempts to transmit the infection by feeding droppings of
infected birds.

Graybill and Smith (1920), Niimi (193?)>

and others failed to produce blackhead when they fed drop­
pings containing free histomonads.

Oral inoculation of

emulsified diseased organs or of cultures have also produced
variable results.

Lund (1955) obtained very low mortality

(1 or 2 percent) in turkeys when he infected them per os
with unprotected histomonads.

He stated that "ingestion of

histomonads along with solids or materials requiring di­
gestion apparently reduced the infectivity of the organisms,
and the ability of the organisms to provoke symptoms was
reduced correspondingly."
infections.

No deaths resulted from these

Horton-Smlth and Long (195&) showed that a low

pH (2.9-3.3) in the gizzards of chickens is lethal to the
unprotected histomonad.

They observed that starved chickens

maintained a higher pH (6 .3-7.0) in the gizzard, thereby
enabling them to infect chickens orally with histomonad
suspensions.

However, the highest incidence of infections

was obtained by administering histomonad suspensions to
starved chickens that had received one gram of alkali mix­
ture (40 percent calcium carbonate, 17 percent magnesium
trisilicate, 43 percent colloidal kaolin) beforehand.

These

investigators also observed the effect of storing histomonad

11
suspensions at different temperatures for varying lengths
of time.

The highest proportion of infections occurred

when the material was administered immediately after its
preparation. At L.*4-°C for 2 hours no infections were pro­
duced.

Tyzzer £t. ai.* (1921) reported that freezing killed

the etiological agent of histomoniasis.
Other methods of transmission of histomoniasis have
been used experimentally.

Tyzzer

(193^) found that rectal

inoculation was more reliable than oral inoculation in pro­
ducing infection.

Farmer and Stephenson (19^9) reported

that rectal inoculation of emulsified ceca produced higher
mortality in turkeys than other methods of infection.

Lund

(1955) attempted to administer quantitative rectal inocula­
tions of histomonad suspensions, and noted that the incidence
of infection, rate of mortality and time of appearance of
symptoms varied with the size of infective dose.

One-

hundred thousand histomonads when introduced rectally into
turkeys resulted in shorter prepatent periods, 100 percent
incidence, and 130 percent mortality.
McGuire and Morehouse (1958) experimentally produced
histomoniasis by transfusing blood from the cecal or mesenteric
veins of diseased donor birds into the wing vein of sus­
ceptible turkeys.

He observed lesions in many of the in­

ternal organs, but cecal Involvement did not occur.

No

infection was produced when he transfused blood from the
heart or wing vein of infected donor birds to susceptible

12
turkeys.

Tyzzer and Fabyan

(1920) produced histomoniasis

in turkeys by the injection of histomonad suspensions subcutaneously and intramuscularly.

Lesions appeared at the

site of inoculation, the histomonad sometimes metastasizing
to the lungs and liver.

Infection of chickens by these

methods proved unsuccessful.

Mechanical transmission of

blackhead experimentally by arthropods has been reported by
Frank (1953)*

Grasshoppers retained viable Heterakls eggs

for at least 96 hours as indicated by Infection of turkeys.
Fifes also were capable of carrying Heterakls eggs mechanic­
ally, but are probably of little importance in natural trans­
mission of the disease.
The lesions and corresponding symptoms of histomoniasis
have been described many times.

The symptoms which appear

are not specific for this disease.

Somnolence, droopy wings

and ruffled plumage, passage of sulfur-colored droppings,
anorexia, emaciation and discoloration of the head are some
of the symptoms that have been observed.

The term '’black­

head” is actually misleading since the blue-black discolora­
tion of the head seldom occurs in Infections with Hlstomonas.
However, due to the common usage of this term, it has been
retained in the literature.

The prepatent period for black­

head varies according to the method of infection.

Follow­

ing the Ingestion of embryonated heterakls eggs symptoms
appear in 11 to 17 d a y s , and mortality occurs in 1^ to 26
days.

Rectal Inoculation shortens the prepatent period

13
and time for mortality to occur by approximately one week.
Many workers have described the gross pathologic conditions,
mainly in reference to the liver and ceca.

Characteristic

liver lesions were described by Lund (1955) as being circum­
scribed (sometimes coalesced), grayish-white
depressed or raised in the center.

in color, and

He described cecal

lesions as consisting of inflammation and ulceration, while
the ceca often contained thick greenish-white material or
large caseous cores.

Peritonitis occurred often.

That the

ceca are the primary site of Infection was shown by Durant
(193°) 9 Delaplane and Stuart (1933) and Schlotthauer £t. al,.
(1933) when they prevented infection with blackhead by
abligatlng the ceca.

Lesions have been produced in other

organs than the liver and ceca.

Farmer

(1951) re­

ported lesions In the spleen, and microscopic examination of
stained tissue sections revealed the presence of Hlstomonas.
Upon subcutaneous inoculation of turkeys secondary lesions
of the lungs, liver, and rarely of the kidneys were observed
by Tyzzer and Fabyan (1920).

Levine (19^7) also reported

blackhead lesions in the kidney, and upon microscopic exam­
ination he was able to observe the histomonad.

Blood in­

duced infections may produce lesions in the lungs, kidneys,
spleer^ pericardium, proventrlculus, and pancreas (McGuire
and Morehouse 1958).

Malewitz and Calhoun (195®) demon­

strated Hlstomonas. microscopically, in lesions of the kidney
and spleen.

They also observed lesions in the lungs,

14
pancreas, and heart, but were unable to demonstrate the
presence of the organism.

Their method of infection utili­

zed embryonated Heterakls eggs.

Using this same method of

Inoculation the author has observed lesions in the liver,
ceca, proventriculus, spleen, mesentery, heart (see Appendix,
plate I), and kidney and Intestinal wall of turkeys and the
ceca, liver, proventriculus

(see Appendix, plate V), and

spleen of chickens.
Microscopic examination of diseased tissues by means
of tissue sectioning and staining has been done by Tyzzer
and Fabyan

(1920), Farmer

house (1958)*

a l . (1951), McGuire and More­

Malewitz and Calhoun (1958)*

general

lesions are characterized by degeneration, necrosis, hemorr­
hage, congestion, serous exudate, lymphocytic infiltration,
and giant cells.
Pathological manifestations in the blood have been
observed by several workers.
reported alterations

Johnson and Lange (1939)

In the differential count and the

development of an anemia in turkeys exposed to Histomonas.
This work was confirmed by McGuire and Cavett (1952) who,
in addition, observed changes in blood glucose (severe hy­
poglycemia occurred in turkeys just before death), erythrocyte
counts and values obtained from the hematocrit.
Factors relating to resistance to infection, Immunity,
and recovery have not been investigated sufficiently.

Curtice

(1907) stated that turkeys may contract histomoniasis at

J-5

any age and that Immunity is not acquired by previous infec­
tion.

Using experiments Tyzzer (1934) described an apparent

development of a resistance to reinfection, following
several rectal inoculations of an attenuated strain of
Hlstomonas.

He suggested that it was necessary to Inoculate

birds when young and that there was a need for constant
reinfection in order to maintain a state of premunition.
He reported that Inoculation of turkeys was less uniformly
successful than the inoculation of chickens, and a loss of
infection included a loss of immunity.

Swales and Frank

(1943) exposed three-, ten-, and seventeen-day-old turkeys
to litter containing embryonated Heterakls eggs.

In most

cases fatal infection was not initiated until the birds were
3 weeks old, and during this time the turkeys did not ac­
quire a low grade infection capable of immunizing them
against fatal histomoniasis later in life.

Sautter e_t a l .

(1950), DeVolt et_ a l . (1954), and McGregor (1951) reported
very little immunity resulting in turkeys when medication,
having a suppressive effect upon the parasite, was removed.
Sautter pt ai,

(op., c l t . ) also noted that whole blood ob­

tained from immune turkeys

(recovered from blackhead) failed

to protect susceptible turkeys.

Waletsky (1950) considered

that there was no certainty that recovered t^rds either with
or without the help of drugs became immune to reinfection.
Lund (1957) reported that rectal inoculation of a nonpathogenlc strain of Hlstomonas failed to protect turkeys

16
3 to 6 weeks later when they were challenged with large
doses of pathogenic histomonads.
vided some protection.

Small challenge doses pro­

He observed that the introduction

of non-pathogenic histomonads per os (via embryonated
Heterqkis eggs) afforded no protection against challenge
doses of the pathogenic strain administered by either route.
Desowitz

(3951)s using rectal Inoculations, showed age to

be a factor Influencing fatal Infections of histomoniasis
in chickens.

Sixty to 71 percent mortality occurred in six

to twenty-one-day-old chickens as compared to 30 percent
mortality in thirty-four-day-old chickens.

On the other

hand, Kendall (1957)* using either embryonated Heterakls
eggs or rectal inoculations of histomonad suspensions, failed
to

observe this age influence In turkeys ranging

from 7days

to 20 months of age.
Serological tests have
Kirkpatrick (1927) and Weldin
3-935)j

been conducted by Rettger and
and Kay (Delaplane

and Stuart

the results were variable and unsatisfactory

for diagnosis of either acute or chronic cases of histomon­
iasis.

MATERIALS AND METHODS
Experimental materials
1.

Chickens and turkeys
All chickens and turkeys used In this investiga­
tion were straight run, and obtained the same day
as they hatched or one day later.

Most of the

chickens were White Leghorns, although some Vantress Cross White Plymouth Rocks were used.

The

majority of the chickens were obtained via parcel
post from H e s s ’s St. Louis Hatchery, St. Louis,
Michigan.

Some were supplied by the Poultry Hus­

bandry Department of Michigan State University.
Two breeds of turkeys were used.

Broad Breasted

Bronze turkeys and some Beltsville Small White
turkeys were supplied by Janssen Farms Hatcheries,
Zeeland, Michigan.

Beltsville Small White turkeys

were also supplied by Wyse Brothers Hatchery, Archbold, O h i o -and the Poultry Husbandry Department of
Michigan State Univers lty.
2.

Feed
Non-medicated turkey starter crumbles (28 percent
protein, antibiotic supplement) and non-medIcated
chick starter mash (20 percent protein, antibiotic

supplement), which were used throughout the investi­
gation, were formulated by the A. E. Staley Manu­
facturing Co., Decatur, Illinois, and obtained
through a local distributor.
Infective materials
Infective materials consisted of embryonated
Heterakls gallinae eggs or cecal washings from tur­
keys suffering from histominiasis.

Some of the

Heterakls eggs were supplied in 0.5 percent formalin
by Dr. Gerald Brody, Moorman Manufacturing Co.,
Quincy, Illinois.

However, the majority of the

infective material was obtained and processed as
follows.

Ceca from chickens, generally over a

year old, were collected several times from poultry
slaughter houses in Linden, Michigan and Middleton,
Michigan.

These ceca were then brought into the

laboratory, slit open, and their contents flushed
into a metal seive suitable for retaining the
mature Heterakls.

Washing was continued until the

worms were clean, after which they were flushed
into a large transparent glass bowl.

Gravid females

were isolated and ground up using a mortar and
pestle.

This material, which contained the un-

embryonated eggs, was suspended in tap water,
centrifuged, and the supernatant discarded.

The

sediment was resuspended in tap water and poured

19
into petri dishes.

Approximately 300 units of

Mycostatlri per ml. of water were added to prevent
the growth of mold.

An embryonation period of 17

days at room temperature was allowed, after which
the suspension was again centrifuged and the super­
natant discarded.

The sediment was diluted in a

flask and the number of embryonated eggs per ml.
determined.

Immediately after shaking the con­

tainer, so as to obtain a uniform distribution of
eg g :5» O.02 ml. of the suspension was transferred
to a glass slide, a cover slip added, and the
number of embryonated eggs counted.

Six of these

counts were made and the mean, multiplied by 5^*
designated the number of embryonated eggs in one
ml.

The embryonated egg suspension was stored at

5° c.
In several instances the infective material con­
sisted of cecal washings (physiological saline)
obtained from clinical cases of blackhead In tur­
keys which had been Infected with 620 embryonated
Heterakls eggs.

This material was then used the

same day as a rectal Inoculum.
A.

Other materials
Fowl pox vaccine

(chick-embryo origin) was ob­

tained from Pitman-Moore Co., Division of Allied
Laboratories, Inc., Indianapolis 6, Indiana.

The

Apothecary Shop, Lansing, Michigan, supplied the
cortisone acetate for intramuscular injection.
Care, management, and distribution of chickens and
turkeys prior to the beginning of experiments
Upon receiving the chickens and turkeys they were
placed in electric brooders which had been operating
one day prior to their arrival.

Here they remained for

3 to U weeks with feed and water supplied &d. libitum.
After 3 to ^ weeks the birds were transferred from the
brooder to wire bottom poultry batteries in separate
rooms (microbiology building and animal disease barn)
according to the grouping in the particular experiment.
Infected and uninfected birds were always retained in
different rooms.
Infection of chickens and turkeys
1.

Per os inoculation
Chickens and turkeys which were to be infected
with Hlstomonas via erabryonated Heterakls eggs
were starved for 16 hours.

The appropriate number

of eggs was introduced directly into the crops of
the birds by means of a one ml. syringe and a rigid
five-inch piece of plastic tube cemented to the
base of a hypodermic needle.

An appropriate number

of eggs was considered to be that number of ernbryonated eggs which when Inoculated per os in turkeys

21
produced 50 percent mortality or greater.

This

number ranged from 300 to ?00 embryonated eggs.
2.

Rectal Inoculation
A fasting period of 16 hours also preceded rectal
inoculations.

Birds were suspended by their legs

and inoculated rectally with 5

of the hlstomonad

suspension prepared from cecal washings of infected
turkeys.

A 5 ml. syringe and a five-inch piece of

number 8 French catheter tubing was used in ad­
ministering the rectal inoculation.

Five ml. of

air was injected immediately following the injec­
tion of the hlstomonad suspension.

These birds

remained in an Inverted position for 30 minutes to
insure a "take" in the infection.
Chickens and turkeys were generally infected by
either method between 3 to 6 weeks of age.
D.

Exposure to various stresses
1.

Starvation
Chickens and turkeys were starved every other
day for 19 days.

Starvation periods, during which

time feed only was withheld, ranged from Zk to 3^
hours with an average of 27*9 hours.

No weights

were recorded, but it was obvious that all birds
were somewhat emaciated at the end of the 19 days.

Simultaneous Elmerla tenella Infections in chickens
Approximately 20,000 sporulated oocysts of Elmerla
tenella were Introduced directly Into the crops of
chickens 6 days after they had received embryonated
Heterakls eggs via the same route.

This number of

sporulated oocysts was selected by infecting chic­
kens beforehand and observing bloody droppings but
no mortality.

Feed was removed several hours be­

fore infection with the coccidia.
Temperature variation
Chickens and turkeys which were to be exposed to
temperatures of k° C. or

C were first trans­

ferred to a twenty-two by fourteen by ten-inch
wire cage (5 birds per cage).

They were then

placed in the walk-in Incubator, maintained at
38° C, or the walk-in refrigerator, maintained at
4° C.

Exposure to these temperatures occurred every

other day for 20 days.

For chickens the exposure

periods ranged from 15 to 19 hours with an average
of 16.6 hours.

Turkeys were exposed from 15 1/2

to 18 1/2 hours or for an average of 16.3 hours.
Aside from leaving several open containers filled
with water in the 38° C incubator no attempt was
made to regulate humidity, but it was measured by
placing a hygrograph on the shelf below the cages
containing the birds.

The hygrograph was first

standardized by placing it in a sealed glass con­
tainer with open dishes of concentrated sulfuric
acid for 11 days and by adjusting the recording
pen to zero on the hygrograph chart.

The humidity

during the exposure of chickens varied from 38
to 65 with an average range of 47.1 to 58.7.

During

the exposure of turkeys the humidity ranged from
33 to 59*5 with an average range of 3 7 *^ to 5^*6 .
All birds exposed to either 4° C or 38° C were
without feed and water during the exposure for the
sake of convenience.
Anemia
Chickens and turkeys were rendered anemic by
periodic withdrawals of blood from either the heart
or the medial wing veins.

Hemoglobin determinations

were made utilizing the acid hematin technique and
the Bausch and Lomb Spectronic 20 colorimeter.
Fowl pox vaccination
Chickens and turkeys were vaccinated with fowl
pox vaccine (chick-embryo origin) by the wing web
(stick) method.

For all vaccinations both wing

webs were pierced.
Cortisone acetate injections
Daily intramuscular injections of cortisone ace­
tate were administered to chickens at a rate of 1.7

to 3.0 mgm. per pound of body weight for 21 days.
Turkeys received daily intramuscular injections
at a rate of 1.7 to 2.5 mgm. per pound for 21 days.
Surgical transplant of cecal material
Cecal contents from 5 protozoan free turkeys, 40 days
old, were collected and suspended in a small quantity
of saline.

This material was then used for surgical

implantation of chicken ceca.

Likewise, cecal material

from 5 protozoan free chickens, 39 days old, was collec­
ted, suspended in a small quantity of saline, and used
for implantation of turkey ceca.
Feed and water were removed from chickens and turkeys
for 24 hours
ate the ceca.

preceding the operation in order to evacu­
Approximately 0.02 ml. of Halatal was

administered intravenously via the medial wing vein.
The anesthesized bird was then secured to a wooden plat­
form, containing several holes, by means of cloth bands.
The area to be Incised was first plucked free of feathers
and swabbed with 70 percent alcohol.

A one-inch incision

was made between the last two left ribs (the same opera­
tion used for caponization).

A surgical spreader held

the wound open while a hooked probe was used to lift
the ceca and intestine, at their point of union, to the
surface.

One ml. of the previously prepared saline sus­

pension of cecal material was injected into each cecum
with a syringe and number 2 0 gauge hypodermic needle.

A small quantity of neomycin sulfate was sprinkled into
the abdominal cavity, and two stitches (cotton thread)
around the rib on either side of the incision were
sufficient to close the wound.

As soon as the birds

recovered from the effects of the Halatal they were
returned to their cages and supplied with feed and water.
For several days after the operation air pockets occurred
under the skin of the turkeys, especially.

Consequently,

the skin was punctured periodically to reduce the
accumulation of air.
All instruments used in the operations were cleaned
in 70 percent alcohol, dried, and flamed

before use.

Serum processing and injection
Whole blood from the appropriate group of birds (usual­
ly 10 to 15 in number) was collected (heart puncture)
and pooled in a sterile flask containing a known amount
of sodium citrate (to give 2 percent sodium citrate by
volume).

The receiving flask was prepared by adding to

it a definite quantity of 10 percent sodium citrate and
placing it in the hot air oven for 2 hours at I500 C.
At the end of this period all water had evaporated leav­
ing a white residue of sodium citrate adhering to the
walls of the flask.

When the desired volume of whole

blood had been obtained, it was immediately centrifuged
for 5 to 10 minutes at 1200 RPM.

The supernatant plasma

was drawn off with bulb pipettes fitted with rubber

suction tubes and transferred Into suitable screwtop
bottles.

The plasma was then heated in a constant

temperature bath at 56° C for 30 minutes, after which
it was cooled and frozen for 2 days.

On the third day

the plasma was thawed and centrifuged in order to remove
the fibrinogen precipitate.

The supernatant serum was

poured off into suitable bottles and used for intra­
venous injection (medial wing veins) the same day.
All materials used in the collection and processing
of blood were sterilized in the hot air sterilizer be­
fore use.
Post mortem examination of chickens and turkeys
All infected and control birds were necropsied as soon
as possible after death.

Carcasses of birds which were

not immediately necropsied were maintained at 4° C
until the autopsy could be performed.

Control birds

and infected birds which did not die from histomoniasis
30 to 35 days after infection were sacrificed by disen­
gaging the cervical vertebrae.

Specimens were observed

for gross lesions from which smears were often made in
order to observe the historaonads.
graded as follows:

Liver lesions were

mild liver involvement or less than

10 lesions, moderate Involvement or 10 to 20 lesions
and severe liver involvement or greater than 20 lesions.
Presence of Hlstomonas in the ceca was determined by
microscopic examination of cecal smears.

Care of tissues for sectioning, and staining methods
Tissues selected for sectioning were cut 5 millimeters
or less in thickness and fixed in 10 percent formalin
for 24 hours.

After the removal of the formalin from

the tissues they were stored in 70 percent alcohol until
they could be dehydrated and embedded in paraffin.
Sections were cut at 6 to 8 u and stained with Harris’
hematoxylin and counterstained with eosin.

Cecal smears

and smears obtained from the various lesions were stained
with May-Greenwald and Giemsa stains.

RESULTS
A.

Surgical implantation of cecal material
Cecal contents were collected from chickens or turkeys,
39 to 40 days old, and prepared for implantation as de­
scribed in the materials and methods.

Birds were in­

fected with embryonated Heterakis eggs, and the results
recorded in table 1 which also contains data from
splenectomized birds.

Chickens, which received cecal

material from turkeys 3 days prior to infection with
Bistomonas. via Heterakis eggs, showed a significantly
lower incidence of cecal lesions and liver lesions of
the lowest grade (less than 10), as compared to in­
fected chickens, which did not receive cecal implantation.
However, the infected, treated birds showed approximately
the same total incidence of liver lesions (sum of three
grades) as infected, untreated birds, and the incidence
of cecal histomonads in both groups was not significantly
different.

One infected chicken (without cecal implan­

tation) possessed lesions in the spleen.

Both groups

of infected turkeys (with or without implantation of
chicken cecal contents) showed nearly the same results
(see table 1).

No lesions cr cecal histomonads were

observed in non-infected chickens and turkeys.
23

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Effect of stress upon histomoniasis in chickens and
turkeys
Starvation of chickens infected with Hlatomonas (via
embryonated Heterakis eggs) apparently had no influence
upon the lesions produced, as indicated by data com­
piled in table 2.

However,

fewer infected and starved

chickens showed Hlstomonas in cecal smears than did
unstarved, Infected chickens*

Turkeys which were

infected and starved showed a higher incidence of liver
lesions (total of three grades), but differences in
occurrence of the three grades of liver lesions were
not significant. Although no weights were recorded,
all starved birds were noticably emaciated at the end
of the starvation periods.

Necropsy of non-infected

birds showed no lesions, and cecal smears were negative
for Histomonas.
Concurrent infections of histomoniasis and coccidiosis
(Elmerla tenella inoculated per o^. 6 days after the
Inoculation of embryonated Heterakis eggs) in chickens
did not significantly alter characteristics of the
hlstomonad infection alone, as indicated in table 3*
No deaths resulted from E. tenella infection alone, but
clinical symptoms (bloody discharges, ruffled plumage
etc.) were observed.

Although 2 of 100 chickens suffer­

ing from both coccldial and hlstomonad infections died,
it was not enough to be of statistical significance.
The data in table 3 was accumulated from two experiments.

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33

No lesions, cecal histomonads or coccidia were observed
in non-lnfected birds.
In another experiment, both chickens and turkeys
infected with embryonated Heterakis eggs were bled
periodically in an attempt to render them anemic.

In­

itial (day of infection), final (day on which survivors
were necropsied), and lowest average hemoglobins and
ranges are recorded in table k-.

Hemoglobin determina­

tions on unbled, non-infected birds were also recorded.
In infected, anemic chickens the lowest average hemo­
globin (6 .9 ) occurred on the ninth day after infection.
The lowest average hemoglobin of Infected, anemic
turkeys fell to 9*7 on the eleventh day after infection.
Corresponding hemoglobins for non-infected, anemic
birds were slightly higher.

Data on mortality, tissue

damage, and Incidence of Hlstomonas in cecal smears
are given in table 5*

No significant variation between

data from infected, anemic and Infected, unbled chickens and
turkeys was noted.

Non-infected birds were negative

for lesions and cecal histomonads.
When chickens and turkeys, Infected with histomoniasis,
were exposed to 4° C or 37° C every other day for 20
days after Infection, variations In mortality, incidence
of lesions, and incidence of Hlstomonas in cecal smears
were negligible (see table 6).

Thirteen of 20 infected

chickens died 16 to 17 days after infection, during

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37

which time they had been exposed to 37° C for 19 hours
(average exposure period, was 16.6 hours).

It Is probable

that these deaths were a result of over exposure, al­
though no mortality occurred in non-infected chickens
exposed for the same length of time at this temper­
ature.

The pathological conditions for both groups

were very similar.

Infected turkeys (not subjected

to temperature exposures) were the same group of
turkeys used in conjunction with the previous experi­
ment.

Necropsy of non-infected birds showed no lesions,

and cecal smears were negative for Hlstomonas.
Table 7 summarizes the data obtained from fowl poxvaccinated or cortisone-treated chickens and turkeys
infected orally with embryonated Heterakis eggs.

No

significant difference was observed between chickens
and turkeys, which received histomonad-containing
Heterakis eggs, and chickens and turkeys, which received
both Heterakis eggs and fowl pox vacination or cortisone
treatment.

A different collection of Heterakis eggs

was used for this experiment, and an increased incidence
of proventriculus and kidney lesions was noted.

In

one turkey, lesions were observed in the proventriculus,
spleen, mesentery, and heart

(see Plate I).

Microscopic

examination revealed the presence of histomonads in
the proventriculus, mesentery

(see Appendix, plates

II and III) and spleen, but none were observed in the

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heart lesion which was characterized by a loose fibrous
thickening of the epicardlum (see Appendix, plate IV),
One vaccinated chicken possessed lesions in the ceca,
liver, and proventriculus (see Appendix, plate V),

Non-

infected chickens and turkeys were negative for lesions
and cecal histomonads.
Immunity and resistance
Chickens, which had been splenectomized and Infected
with Hlstomonas (via embryonated Heterakis eggs), sur­
vived and showed no significant difference in tissue
destruction or Incidence of Hlstomonas in cecal smears,
when compared with non-splenectoraized chickens suffer­
ing from blackhead (see table 1).
In order to determine if host age influences resistance
mortality, incidence of lesions and incidence of Hlstom­
onas in cecal smears were observed in chickens and
turkeys of different ages (see table 8).

No mortality

was observed in 162 chickens ranging in ages from 18
to 131 days.

The incidence of cecal and liver lesions

and cecal histomonads was consistent.

Turkeys of all

age groups, ranging from 10 to 62 days, suffered from
40 to 100 percent mortality losses (see table 8).

Out

of a total of 142 turkeys, representing all age groups,
97 or 68.3 percent died.

No age group of turkeys de­

monstrated a resistance to histomoniasis, as Indicated
by the mortality and tissue damage.

The incidence of

40
TABLE 8
HISTOMONIASIS IN CHICKENS* AND TURKEYS** OF DIFFERENT AGES
Age
in
Days

N o . of
Birds
per
Group

Chickens
18
6
21
20
20
22
8
25
27
29
28
20
32
5
40
29
42
15
56
131

No. of Birds with Lesions
No. of
KidDeaths Ceca Liver Spleen ney

Prov

No. of
Birds
Containing
Hlstomonas

0
0
0
0
0
0
0
0
0
0
0

0
5
5
1
5
3
2
10
8
5
5

1
2
1
1
8
6
3
14
5
5
5

0
0
0
0
0
0
0
1
1
0
0

0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0

5
15
12
6
26
18
3
27
11
5
5

5
4
22
4
25
20
10
5
7
15
25

2
2
13
4
20
10
5
2
5
10
24

2
2
18
4
23
14
6
2
6
11
24

2
2
15
4
22
13
6
2
6
11
25

0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
2
0
0
0
0
0
1

0
0
3
0
6
0
0
0
0
0
5

2
3
21
4
25
19
7
4
7
12
25

Totals
Chic­
162
kens
Turkeys 142

0
97

49
102

51
108

2
0

0
3

0
14

133
129

Turkeys
10
17
21
25
29
31
35
36
42
43
62

*Represents a majority of White Leghorns and some Vantress
Cross White Plymouth Rocks.
**Represents a majority of Beltsvllle Small Whites and
some Broad Breasted Bronze.
***Sacrificed 17 days after infection.

41
cecal histomonads was high in both chickens and turkeys
(82 and 91 percent, respectively), thus proving a high
degree of experimental exposure to the hlstomonad.

The

data accumulated in table 8 represents infections in
Vantress Cross White Plymouth Rock and White Leghorn
chickens and Beltsville Small White and Broad Breasted
Bronze turkeys.
One or 5 inoculations of embryonated Heterakis eggs
were administered orally to two groups of chickens in
order to observe the effects of challenging doses upon
the course of the disease.

Surviving birds which re­

ceived only one oral inoculation were sacrificed JO
days later, whereas surviving birds which received 5
oral inoculations (two-week intervals) were sacrificed
30 days after the fifth inoculation (60 days after the
first inoculation).
tained in table 9 *

Data for this experiment are con­
No mortality occurred in either

group (1 or 5 inoculations), and pathological conditions
were consistent except for the incidence of liver lesions.
Nine of 24 chickens, receiving a single inoculation,
possessed less than 10 liver lesions, and 3 chickens
had 10 to 20 lesions in the liver.

Only 4 of 24 chickens,

receiving multiple inoculations had less than 10 liver
lesions, and more severe liver lesions were not observed
in this group.

Although these variations In grades of

liver lesions were not very large, the difference between

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43

the total number of birds possessing any grade of liver
lesions (total incidence) was statistically significant*
One chicken was observed with lesions in the spleen.
Turkeys were also
Heterakis eggs

inoculated orally with embryonated

and challenged at two-week Intervals.

However, in this case the first two inocula administered
to turkeys which were to receive multiple doses, con­
tained an insufficient number of histomonads to produce
mortality.

Succeeding inocula, obtained from a new

collection of Heterakis were sufficient to produce
mortality, as indicated when 24 of 25 turkeys, which
received one inoculation, died.

Eighteen of 25 turkeys,

which received 5 inoculations (last 3 inoculations suf­
ficient to produce mortality) died of histomoniasis.
As can be seen from the results in table 9> the incidence
of severe liver lesions (greater than 20) was less for
turkeys receiving multiple inoculations.

The occurrence

of any grade of liver lesions (total), however, was
nearly the same for both groups, since 6 of the birds
receiving multiple inoculations had less than 20 liver
lesions.
Table 9 also contains data obtained from infected
turkeys (one inoculation sufficient to produce mortality),
each of which received an intravenous Injection of serum
collected from chickens that had recovered from his­
tomoniasis.

In this group of turkeys mortality, incidence

44
of cecal lesions and severe liver lesions, total of three
grades of liver lesions, and incidence of proventriculus
lesions were significantly lower than those values ob­
tained from infected turkeys not receiving chicken serum.
Only 3 oiAt of the 20 infected, serum treated turkeys had
died after 22 days.

Thirty-two days after infection,

at which time survivors were generally autopsied, black­
head symptoms were again observed, and 3 to 5 days later
5 more turkeys died.

Since turkeys were injected with

serum 3 days after infection, a period of J 2 days had
elapsed between the serum injection and the second
occurrence of fatal blackhead.

Forty-two days after

infection no further symptoms were observed and the 12
surviving birds were necropsied.

In like manner, in­

fected chickens were injected intravenously with serum
obtained from turkeys that had recovered from blackhead.
The occurrence of cecal, liver, and spleen lesions and
cecal histomonads was approximately the same for both
infected groups, either with or without serum injection.
No blackhead lesions were observed in non-infected birds.
The next experiment was, in part, a repetition of the
previous one (infected turkeys received immune chicken
serum).

Non-lraraune serum was collected from chickens

which had been reared in a separate room and which had
no contact with histomoniasis.

Three groups of 22

turkeys were Infected orally with Hlstomonas (via

^5
embryonated Heterakis eggs).

One group received Immune

chicken serum four days after Infection, another group
i

received non-immune chicken serum four days after In­
fection, and the third group received no serum at all.
Three groups of 8 turkeys were treated in the same manner,
only they had been infected rectally with a suspension of
hlstomonads.

The injection of Immune chicken serum into

orally infected turkeys decreased

significantly

severity of histomoniasis (see table 10),

the

Lower mor­

tality and Incidence of cecal lesions and severe liver
lesions were observed in serum-treated turkeys than in
untreated turkeys; however, the total incidence of the
mild, moderate, and severe liver lesions in each group
was not significantly different since most of the liver
lesions in serum-treated turkeys were mild or moderate,
whereas most of the liver lesions in untreated turkeys
were severe.

On the other hand, orally infected turkeys

treated with non-immune chicken serum showed approxi­
mately the same mortality and incidence of blackhead
lesions and cecal histomonads as did orally infected
turkeys not injected with serum.

One-hundred percent

mortality occurred in rectally infected turkeys of all
three groups, although it did not occur at the same time.
Rectally infected turkeys, given immune or non-immune
serum or no serum at all, suffered 100 percent losses in
16, 14, and 12 days, respectively.
observed in non-infected turkeys.

No lesions were

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DISCUSSION
Diseases in poultry may range from little host
specificity to species specificity*

Certain diseases (often

of viral etiology) terminate fatally in both chickens and
turkeys), whereas others may affect chickens more severely
than turkeys or vice versa.
specific (coccldiosis).

Still other diseases are host

Poultry diseases of protozoan

etiology generally fall under the last two categories.
Histomoniasis, as indicated in the review of literature,
affects turkeys more severely than chickens.

From this

aspect the chicken might be considered a more normal host.
Since fatal blackhead does occur in chickens under field
conditions, an attempt was made in this investigation to
determine what factors are responsible for this fatal
termination, and also what factors provide the chicken with
a greater resistance to the disease.
At the beginning of this investigation it was often
observed that cecal histomonads in stained preparations
from chickens and turkeys showed the presence of bacteria
within their cytoplasm.

A review of the literature indicated

that Hlstomonas has not been cultured la. vitro in the ab­
sence of bacteria.

The disease, itself, is initiated in

the ceca where there is an abundance of bacteria.
4?

In view

48
of these facts surgical implantation of cecal contents from
turkeys to chickens, and vice versa, was performed in order
to determine whether bacterial flora might affect the di­
sease.

From results in table 1 it appears as though im­

plantation of turkey cecal contents Into ceca of chickens
effected a decrease In the incidence of cecal lesions and
mild liver lesions (less than 10) as compared with the
incidence of these lesions In Infected, untreated chickens.
Therefore,the Incorporation

of turkey cecal contents into

ceca of chickens definitely did not result in conditions
favorable for progressive histomoniasis. Implantation

of

chicken cecal contents into ceca of turkeys was without
effect upon histomoniasis in this host.
It is the opinion of some workers that fatal histo­
moniasis occurs in chickens when their resistance has been
lowered by vaccination, other diseases, or some adverse
condition.

In the present investigation several stresses

were used in an attempt to break down the resistance of
chickens or alter the susceptibility of turkeys to black­
head.

Periodic starvation of birds tc the point of emaci­

ation did not suffice to lower the resistance of chickens
sufficiently to produce mortality due to histomoniasis,
although It did decrease the incidence or cecal histomonads
(see table 2).

Although there was no significant change In

mortality, the disease in starved turkeys resulted in a
higher incidence of cecal

lesions.

Therefore, the indications

are that starvation may have had some Influence upon the
disease in turkeys (increased liver lesions), but no in­
fluence upon the disease in chickens.
An induced, anemic condition was not sufficient to
alter histomoniasis in either chickens or turkeys, as indi­
cated in table 5*

A slight anemia resulted from hlstomonias

in chickens and turkeys, as indicated by the comparison of
hemoglobins of infected and non-infected birds which were
bled periodically (see table 4).

That a slight anemia

develops in Infected turkeys has been demonstrated by
Johnson and Lange (1939) and McGuire and Cavett (1952).
Likewise, the repeated exposure of Infected chickens and
turkeys to 4° C or 37° C did not change the mortality or
pathological conditions characteristic of the disease (see
table 6).
Under field conditions when two diseases such as
coccidiosis and histomoniasis occur simultaneously it is
difficult to determine whether one influences the other.
However, under experimental conditions, more accurate in­
formation can be obtained.

Chickens were infected with

just enough sporulated oocysts of Elmeria tenella to pro­
duce clinical symptoms, but not mortality.

The results in

table 3 indicate that coccidiosis did not significantly
increase mortality or the incidence of lesions due to
histomoniasis.
of Jowett

These results do not support the belief

(1911) that a disruption of the intact epithelium

50

by coccidia aids the invasion of Histomonas.
Lund (1956) has stated that vaccination may lower
the resistance of chickens thereby allowing them to con­
tract fatal histomoniasis.

Results obtained in the present

Investigation, using fowl pox vaccination, disagree with
L u n d ’s belief.

Infected and vaccinated chickens and turkeys

failed to show any significant change in mortality or
incidence of lesions and cecal histornonads when compared
with Infected, unvaccinated chickens and turkeys (see table
7).
Since none of the afore-mentioned stresses were
capable of Inducing fatal histomoniasis in chickens or in­
fluencing mortality in turkeys, the Investigation was con­
tinued along the lines of natural or acquired resistance.
Kendall (1957) infected turkeys of different ages (7 days
to 20 months) orally with embryonated Heterakls eggs or
rectally with histomonad suspensions, and observed uniform
susceptibility to histomoniasis.

On the other hand, Desowitz

(1951) infected chickens of different ages (6 to 3^ days)
rectally with suspensions of histornonads, and he observed
less mortality (30 percent) in thirty-four-day-old chickens
than in six to twenty-one-day-old chickens (60 to 71 percent
mortality).

However, this method of infection is unnatural,

and the number

of histornonads introduced into the ceca

was probably much greater than the number which would be
introduced orally by means of embryonated Heterakls eggs.

51

In the present work data were accumulated from many experi­
ments in which all Infections were produced by the oral
inoculation

of embryonated Heterakls eggs.

One hundred

and sixty-two chickens ranging In ages from 18 to 131 days
showed uniform resistance to histomoniasis (see table 8).
Pathological conditions occurred with uniform frequency,
and chickens in all age groups recovered.

The greater

proportion of these chickens (133 of 1 6 2 ) retained histornonads
within their ceca for at least 30 to 42 days after infection,
at which time they were necrcpsied.

Turkeys, on the other

hand, showed uniform susceptibility to histomoniasis (see
table 8).

Out of 142 turkeys 97 or 68.3 percent died of

histomoniasis.

Mortality for the different age groups

(ranging from 10 to 62 days) varied from 40 to 100 percent.
These results are in agreement with Kendall’s (pp.. clt. ) .
namely, that turkeys of all ages are susceptible to his­
tomoniasis.
It was apparent that under experimental conditions
a percentage of turkeys acquired an infection which was
relatively non-pathogenic since the Incidence of lesions
and cecal histornonads was higher than the occurrence of
mortality (see table 8).

Resistance to succeeding pathogenic

challenge doses may indicate a state of premunition in
turkeys, as reported by Tyzzer (193^)*

Other workers have

also noted the existance of a temporary resistance to rein­
fection.

Results of the present Investigation, listed in

52
table 9, agree with results obtained by other workers.
Turkeys were infected with embryonated Heterakls eggs five
times (two week intervals).

The first two Inoculations

were sub-lethal, although symptoms did appear.

Succeeding

challenge doses were lethal, as Indicated when 24 of 25
turkeys, that had not received an inoculation previously,
died as a result of one inoculation.

Turkeys which received

multiple Inoculations suffered mortality losses after each
of the last three inoculations.

However, these turkeys

showed less mortality and a lower incidence of severe
liver lesions when compared with turkeys receiving a single
inoculation (see table 9).

Statistical analysis showed

the differences to be significant at the 5 percent confidence
level.

In reference to these results it can be said that

turkeys may possess some resistance to reinfection, but
this resistance is by no means complete, since mortal­
ity did occur after repeated Inoculations.

Chickens, which

received a total of five pathogenic Inoculations at inter­
vals of 2 weeks, showed a lower total incidence (significant
at the 5 percent confidence level) of liver lesions when
compared writh chickens that had received one inoculation.
Here again the indications are that a resistance to rein­
fection exists, only in this case the initial resistance
is greater at the first oral inoculation since no mortality
occurred.

Subsequent Infections, though, did not increase

the pathological conditions characteristic of the disease.

53

That turkeys, which recover from histomoniasis,
possess an incomplete and temporary immunity has been point­
ed out previously in the review of the literature.

It has

been demonstrated by Sautter and co-workers (1950) that
whole blood , obtained from turkeys which had recovered from
blackhead, failed to protect susceptible turkeys from the
fatal disease.

To the author’s knowledge this has not been

tried in chickens suffering from histomoniasis, or for that
matter, blood from Infected, recovered chickens has not been
injected into Infected turkeys.

In the present investi­

gation serum was obtained from chickens and turkeys that
had recovered from histomoniasis.

The turkey serum was

Injected into chickens 3 days after infection with Heterakls
eggs, and the results in table 9 Indicate that the serum
had no effect upon the disease in chickens.
serum from control turkeys

For this reason

(no contact with blackhead) was

not injected into infected chickens.

On the other hand,

when serum from recovered chickens was injected into turkeys
3 days after infection with Heterakls eggs, an Impressive
change in the course of the disease was noted (see table 9)Mortality was decreased considerably and lesions were less
severe and occurred less frequently.

3y 32 days after the

oral inoculation of turkeys with embryonated Heterakls eggs
(29 days after serum injection) only 3 of 20 turkeys had
died, and they showed typical histomonad lesions.

In pre­

vious experiments surviving birds were necropsied at this

length of time after Infection, but symptoms of histomoniasi
were again observed. (32 days after infection), and necropsy
was delayed for 10 days, during which time 5 more deaths
occurred and symptoms of blackhead disappeared*

The occur­

rence of the last 3 deaths was not attributed to reinfection
by the ingestion of food or water that had been contaminated
with Heterakls eggs passed by gravid worms in the turkeys,
since there was insufficient time for the worms to mature,
the eggs to embryonate, and turkeys to become infected by
ingesting the embryonated eggs.

Nor were these deaths

attributed to infection by the ingestion of free histornonads
in the feces since all turkeys were retained in batteries
with wire bottoms.

Therefore, it appears that the chicken

serum afforded the last 5 turkeys that died protection from
histomoniasis for approximately 3 weeks.

By 32 days after

the infection of a similar group of turkeys which had re­
ceived. no serum injection, 2b of 25 had died.

All of these

turkeys, except the lone survivor, which possessed less
than 10 liver lesions and no cecal lesions, showed very
severe liver and cecal lesions.

Five of the 2b birds which

had died also showed grayish necrotic lesions on the wall
of the proventriculus (see Appendix, plate I).

The lesions

always occurred on that portion of the proventriculus which
remained in contact with the highly necrotic liver, and
they gradually spread from that area.

It Is the author's

opinion that such lesions were not initiated by iiistornonads

55

from the blood stream, but that a direct transfer of the
histornonads from the liver (through the disrupted liver
capsule) to the wall of the proventriculus initiated the
lesions.

Previously, McGuire and M0rehouse (195(
5) reported

proventriculus lesions in one turkey that had been infected
Intravenously (blood inoculum derived from cecal or mesen­
teric veins of infected donor), but they did not state
whether or not the lesions resulted from histornonads in
the blood stream.

That the proventriculus lesions resulted

from a direct transfer of histornonads from the liver is
supported by the observation in one turkey of a similar
lesion on the wall of the small intestine where it lay in
contact with the necrotic liver.

Microscopic examination

of proventriculus sections revealed coagulation necrosis
and inflammation, and the histornonads were observed in
abundance

(see Appendix, Plate III).

Fresh saline smears

also revealed the presence of the organisms.

None

of

the turkeys which had received serum injections showed
lesions in the proventriculus.
At this stage of the investigation It remained to
be determined whether the serum obtained from recovered
chickens contained Immune bodies or whether the protection
afforded turkeys was derived from some inherent property
of chicken serum.

Therefore, serum was collected from

chickens which had no contact with histomoniasis, and from
other chickens which had been infected for 21 days.

The

ability of serum, obtained from infected, recovered chickens,
to protect turkeys from the progressive form of the disease
was confirmed, as indicated

by data in table 10.

Turkeys,

which were infected with Hlstomonas and injected with nonimmune chicken sera, experienced no protection against the
disease.

Mortality and Incidence of lesions were not

significantly different from the results obtained from in­
fected turkeys which received no serum injection.

This

would seem to Indicate the existance of humoral antibody in
recovered chickens.

Three groups of 8 turkeys were infected

rectally with histomonad suspensions, the results of which
are shown in table 10.

One group received immune chicken

serum, another group received non-immune chicken serum, and
the third group received no serum.

All three groups of

turkeys died, but 100 percent mortality of turkeys receiv­
ing Immune serum occurred later than it did In the other
two groups, thereby suggesting the benefit of some protection
derived from the serum.
Since the information on the ability of Immune chicken
serum to afford some protection to turkeys from histomoniasis
came late in this Investigation, time did not permit suf­
ficient supportive experimentation on antibody production.
Five chickens were splenectomized and inoculated per os
with embryonated Heterakls. eggs.

This operation was not

sufficient to induce fatal histomoniasis; nor did the
Incidence of lesions and cecal histornonads differ significant

57

from results obtained from non-splenectomized, Infected
chickens

(see table 1 ).

Bjorneboe and co-workers (1951) had shown that cor­
tisone may affect the concentration of humoral antibody, so
chickens and turkeys were Inoculated orally with embryonated
Heterakls eggs and treated with daily Intramuscular Injec­
tions of this hormone for 21 days after infection.

Fullin

(1955) injected turkeys Intramuscularly with 1.5 mg®* of
cortisone per pound, since this was the suggested dosage
for rheumatoid arthritis In man.

He gave daily injections

for 19 days and observed no toxic effects in turkeys re­
sulting from the cortisone.

Since there was no toxic

effects at this dosage, an Increased dosage was used In
the present investigation.

Chickens received 1.7 mgm. per

pound of body weight for the first 5 days, and the dosage
was gradually increased to 3*°0 mgm, per pound by the tenth
to thirteenth day, after which it was gradually decreased
on successive days to 2.33 mgm. per pound.

Turkeys also

received 1.7 mgm. per pound for the first 5 ^ays, after
which the dosage was gradually Increased daily to 2.^3 mgm.
per pound on the nineteenth to twenty-first day.

Pullin

(o p . clt.) observed no effect of cortisone upon histomoniasis
in turkeys, but he had not used chickens in his experiments.
In the present investigation Pullin's results were confirmed,
and daily cortisone injections in chickens also failed to
alter the disease significantly (see table 7).

Since

53

cortisone has been shown to decrease antibody concentration
and Inflammatory reactions, one might expect the severity
of histomoniasis in chickens and turkeys to increase.

How­

ever, this was not observed, possibly due to Insufficient
dosage.
An insight to the exact cause of death of turkeys
suffering from histomoniasis might shed some light upon
fatal histomoniasis in chickens.

McGuire and Cavett (1952)

observed the development of a severe hypoglycemia in turkeys
just before they died of blackhead, and they stated that
this factor might be the cause of death.

From time to time

during the present investigation when turkeys were observed
a few hours away from death intravenous Injections of 5
percent glucose (5 to 15 m l . ) were administered In an
attempt to prolong the life of the bird, but to no avail.
On another occasion 6 turkeys were inoculated rectally
with histomonad suspensions, and on each day thereafter b
ml. of a 5 percent glucose solution was Injected intraven­
ously as long as the turkeys lived.

From the fifth day

after Infection (symptoms appeared) until death the turkeys
were also injected Intramuscularly with 2 ml. of a 5 percent
glucose solution.

The injections of glucose did not pro­

long the lives of turkeys infected with histomoniasis.

These

results Indicate that the hypoglycemia which develops may
not be severe enough to cause death of turkeys.

SUMMARY
Surgical implantation of cecal contents from non­
infected control chickens into ceca of turkeys, followed by
infection with embryonated Heterakls eggs, failed to alter
the course of the disease significantly In turkeys.

The

surgical implantation of cecal contents from non-infected
control turkeys into ceca of chickens, followed by Histomonas
infection, failed to produce conditions favorable for pro­
gressive histomoniasis.

Infected chickens receiving Implants

showed a lower incidence of cecal lesions and of mild liver
lesions, possibly indicating the creation of an unfavorable
cecal environment.
Starvation of chickens suffering from histomoniasis
did not increase the severity of the disease.

A signifi­

cantly lower incidence of cecal histornonads was observed.
Starvation of turkeys suffering from histomoniasis increased
significantly the incidence of liver lesions, but did not
affect mortality.
Concurrent infections of histomoniasis and coccidiosis
in chickens did not increase significantly mortality and
incidence of lesions due to histomoniasis.
Rendering chickens and turkeys anemic did not change
the pathogenesis of histomoniasis as it generally occurs
59

6o

in these hosts*

Slight anemias in chickens and turkeys

were developed as a result of the disease alone.
Exposure of chickens and turkeys to b° C or 37° C
temperatures failed to influence any aspect of the disease
in these hosts.

Likewise, the vaccination of chickens and

turkeys with fowl pox virus failed to change significantly
mortality,

incidence of cecal histornonads, or incidence of

les ions•
Turkeys of all age groups
to be equal

(10 to 6k cays) were found

susceptible to histomoniasis when inoculated

per os with embryonated Heterakls eggs.

Chickens of all age

groups (18 to 131 days), on the other hand, were found to
be equally resistant to the disease when inoculated with
embryonated Heterakls eggs.
with uniform frequency.

Pathological conditions occurred

Chickens in all age groups recovered,

while turkeys suffered mortality losses from bo to 100 per­
cent .
Turkeys, which received 2 sub-lethal oral inoculations
of embryonated Heterakls eggs, followed by 3 lethal inocu­
lations (two-week intervals), showed less mortality and a
lower incidence cf severe liver lesions (greater than 20
lesions), thereby indicating some resistance to reinfection.
Mortality occurred, however, after each lethal dose.

Chic­

kens, which received 5 lethal oral inoculations of embryonated
Heterakls eggs

(lethal for turkeys), showed a decrease in

the incidence of liver lesions (any grade), again indicating

61

some resistance to reinfection.

Ho mortality vias observed

in these chickens.
Serum, collected and pooled from turkeys (20 to 21
days after oral inoculation with embryonated Heterakls eggs),
and injected into chickens

(3 to b days after oral inocula­

tion of embryonated Heterakls eggs), had no effect upon
histomoniasis in chickens.

In like manner, serum, collected

and pooled from uninfected control chickens, when injected
into turkeys had no significant effect upon the pathogenesis
of histomoniasis.

However, when serum was collected and

pooled from chickens (20 to 21 days after per os inoculation
with embryonated Heterakis eggs), and injected into turkeys
(3 to b days after infection), mortality decreased signifi­
cantly.

Corresponding significant decreases occurred in

the incidence of cecal,liver, and proventriculus lesions.
Immune chicken serum afforded less protection (delayed 100
percent mortality) to turkeys when they were Infected
rectally with histomonad suspensions.
Splenectomy, followed by oral Inoculation of embryonated
Heterakls eggs, had no effect upon hitomoniasis in chickens.
Chickens and turkeys, which received daily intra­
muscular injections of cortisone for 21 days after oral
inoculation of Hlstomonas. via Heterakls eggs, showed no
significant change in mortality and incidence of lesions.
Injection of a 5 percent glucose solution into
turkeys just before death or daily Injections after the

62

time of

infection until death did not prolong the lives of

turkeys suffering from histomoniasis.
An increased Incidence of lesions on the wall of
the proventriculus next to the liver occurred when ground
up Heterakls females from the last two collections (from
poultry abattoirs) were used as the source of inoculum.

It

appears that these lesions are not initiated by histornonads
from the blood stream, but by a direct contingent migration
from the liver, through the disrupted
wall ofthe proventriculus.

liver capsule, to the

In one turkey an

Identical lesion

occurred on the wall of the small intestine where it lay
in contact with the necrotic liver.

Microscopic examination

of tissue sections revealed coagulation necrosis and in­
flammation of the muscular layers and the presence of many
histornonads•
Lesions resulting from histomoniasis, alone or in
conjunction with a particular treatment, have been observed
in chickens (^78 infected) in the following organs (the
number in parentheses indicates the number of observations):
ceca (9^), liver (130), spleen (3 ), and proventriculus (1 ).
Histornonads have been observed in these lesions.

In like

manner, the following lesions have been observed in turkeys
(355

infected) suffering from histomoniasis:

liver (256),

ceca (253), proventriculus (3 6 ), kidney (8 ), spleen (3 ),
intestinal wall (1), mesentery (1), and heart (1 ).

His-

tomonads have been observed in these lesions with the

63
exception of the heart.
The incidence of Hlstomonas in cecal smears from
experimental chickens and turkeys Infected with 300 to 7^°
embryonated Heterakls eggs has been high throughout the
investigation, indicating a high degree of exposure of
birds to the etiological agent.

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67
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68

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APPENDIX

PLATE I

Lesions due to histomoniasis in a turkey. Note
lesions in the liver, ceca, proventriculus, spleen,
mesentery, and one lesion on the heart.

PLATE I.

71

PLATE II.

Heart of a turkey infected with histomoniasis.
Notice the thick fibrous layer on the epicardium.

72
PLATE II.

PLATE III.

Froventrlculus of a turkey infected with
histomoniasis.

73

PLATE III,

PLATE IV.

Mesentery of a turkey infected with histomonlasis.
The thickened area of the mesentery shows many
small, rounded histomcnads.

PLATE IV.

7^

PLATE V,

Lesions due to histomoniasis in a chicken. Note
lesions in the liver, ceca, and proventriculus.

PLATE V,

75

OBSERVATIONS ON SOME FACTORS WHICH MAY INFLUENCE
SUSCEPTIBILITY OR RESISTANCE OF CHICKENS AND TURKEYS TO
HISTOMQNIASIS

By
CLARENCE JOSEPH WELTER

AN ABSTRACT
Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health
1959
o

Approved b y :

__

CLARENCE JOSEPH WELTER
ABSTRACT
A study was made concerning factors which may in­
fluence susceptibility or resistance of chickens and turkeys
to histomoniasis.

These factors included surgical implanta­

tion of cecal contents from chickens to turkeys and vice
versa, exposure to various stresses

(starvation, Elmerla

tenella infections In chickens, anemia, temperature variation,
cortisone Injection, fowl pox vaccination), and the deter­
mination of the effect of age, multiple inoculations, and
immune or non-Immune serum injections.
Most of the birds were infected orally with 3 00 to
700 embryonated Heterakls eggs (sufficient to produce 50
percent or greater mortality in turkeys).
rectally with hlstomonad suspensions.

Some were infected

Surviving birds

were necropsied 30 to 35 bays after infection.
Surgical implantation of cecal material failed to
alter histomoniasis in chickens and turkeys with the ex­
ception of a decreased incidence of cecal and mild liver
lesions in chickens.
Starvation of infected chickens and turkeys did not
affect mortality, but turkeys showed a significant Increase
in the incidence of liver lesions.
Concurrent Infections of histomoniasis and coccidiosis
in chickens did not significantly change mortality and inci­
dence of lesions due to histomoniasis.

CLARENCE JOSEPH V/ELTER

2

Rendering chickens and turkeys anemic or exposing
them to 4° C or 37° C temperatures or vaccinating them had
no significant effect upon histomoniasis in these hosts.
Turkeys of all age groups (10 to 62 days) were found
to be equally susceptible to histomoniasis, and chickens
of all age groups (18 to 131 days) were equally resistant
to the disease.

All chickens recovered, while turkeys

suffered from 40 to 100 percent mortality losses.
Two sub-lethal oral inoculations of Hlstomonas (via
embryonated Heterakls eggs), followed by three lethal oral
doses (at two-week intervals), had the effect of decreasing
mortality and incidence of liver lesions in turkeys.

Ne­

cropsy of chickens after five lethal Inoculations (lethal
for turkeys) revealed a lower incidence of liver lesions,
Turkey serum (collected 20 to 21 days after oral
Inoculation with embryonated Heterakls eggs) when injected
into chickens had no effect upon histomoniasis.

In like

manner, serum, collected from non-infected chickens, when
injected into turkeys did not alter the disease.

However,

when serum was collected from chickens (20 to 21 days after
per os inoculation with embryonated Heterakls eggs) and
Injected into turkeys, mortality due to histomoniasis de­
creased considerably.

In two separate experiments corres­

ponding significant (5 percent confidence level) decreases
occurred in the incidence of cecal and liver lesions.
one experiment a significant decrease In occurrence of

In

CLARENCE JOSEPH WELTER
proventrlculus lesions was observed.

3
Immune chicken serum

from this same collection afforded less protection (delayed
100 percent mortality) to turkeys when they were infected
rectally with histomonad suspensions.
Splenectomy had no effect upon histomoniasis in
chickens.

Likewise, infected chickens and turkeys, which

received daily Intramuscular injections of cortisone for
21 days, showed no significant change in mortality and
Incidence of lesions.
Injections (just before death or daily until death)
of 5 percent glucose solution into turkeys did not prolong
the lives of turkeys suffering from histomoniasis.
Lesions resulting from histomoniasis have been
observed in the ceca, liver, spleen and proventrlculus of
chickens and the liver, ceca, proventrlculus, kidney,
spleen, intestinal wall, mesentery and heart of turkeys.
Histomonads were observed in these lesions with the exception
of the heart.