PATHOGENESlS 0F
ACUTE MEAN MALARIA

Thesis for the Degree of 3312 2‘)
MICHEGAN STATE iifiiVERSiW
HYA L. SEMI

’ 1973'

'3‘? 'dfl'i" ‘,..... ...’o.r 4
air

LIBRA f- 1
Michigan State

University;

nx‘zr- I1:

This is to certify that the

thesis entitled

PATHOGENESIS OF ACUTE AVIAN MALARIA

presented by
Jiya L. Soni

has been accepted towards fulfillment
of the requirements for

Ph -D - degree in Microbiology

{4 1&1 1;; Z 1_

Major professor

Date _A1_1.gll_8t_i,_l_913_

0-7 639

 

 
   
  

BIN‘DINGBYV
1 111111311an3 .
1.311011111110111 1111‘ 1

11157R91YBlN-5» 1

   

1
1

11:1-

 

ABSTRACT

PATHOGENESIS OF ACUTE AVIAN MALARIA

By

Jiya L. Soni

Factors in the plasma of chickens with acute PZasmodium gaZZinaceum
infection were found to be responsible for both anemia and acute glomeru-
lonephritis. Intravenous injection of plasma of malarious birds into
normal chickens produced a 35% reduction in the red blood cell counts
within 48 hours which persisted for 8 to 9 days. A similar anemia
resulted from the injection of material eluted from cells of malarious
blood. Anemia was also induced by injections of a cold-active agglutinin
absorbed from the plasma of malarious chickens with trypsinized human
type "0" erythrocytes and dissociated from the cells by incubation at 37
C. The injection of the agglutinin also produced an anaphylactic—like
shock in the recipients, but no deaths. When the plasma that had been
absorbed free of agglutinin was injected into normal chickens it too
produced anemia. Thus there appeared to be 2 substances in malarious
blood that caused anemia.

Acute glomerulonephritis was produced in normal chickens by the
injection of plasma from malarious birds. Except that it appeared much
earlier, this nephritis did not differ from that seen in chickens with
acute P. gallinaceum infection. Since the plasma did not cause nephritis

in chickens that had recovered from acute P. gallinaceum infection, it

Jiya L. Soni
was considered that the inducing substances were immunologic rather than
host permeability factors.

Study of the blood of malarious chickens revealed that the plasma
contained the cold-active hemagglutinin, globulin associated antigen
(serum antigen), antibody to the antigen, and antibody to extracted
parasite antigen. Material leached from cells of malarious blood con-
tained predominantly serum antigen and antibody that did not differ from
that found in the plasma. The parasites liberated from washed disrupted
erythrocytes revealed the presence of one or more antigens that were not
differentiated one from the other. These antigens did not react with
antibody to the serum antigen but did react with the antibody to para-
site antigen in plasma of malarious birds.

Study of the cold-active agglutinin revealed that it was associated
with the beta globulin fraction of the plasma, was inactivated by 2-
mercaptoethanol cleavage, and reacted with red blood cells, and anti-
serum to whole chicken globulin. Cells of chickens that had been
injected with the cold agglutinin reacted with fluorescein isothio-
cyanate conjugated anti-chicken globulin, but did not react with conjur
gates of antibody to serum antigen or antibody to parasite antigen.

Both serum antigen and antibody to serum antigen were extracted
from the kidneys of malarious chickens and both were identified and
partially separated from the various serum proteins that were found in
the urinary droppings of malarious chickens. As birds recovered from
acute infection, only the antibody and low molecular weight protein
continued to be excreted.

When kidney sections from malarious chickens were reacted with
conjugates of antibody to serum antigen, antibody to parasite antigen,

and anti-IgG of chickens, immunofluorescent reactions were seen with

Jiya L. Soni
each; however, the reactions of the glomeruli with the first 2 conju-
gates was stronger, more diffuse and granular than was the reaction
with anti-parasite antigen. Kidney sections of chickens with nephritis
induced by plasma injections reacted with conjugates of antibody to
serum antigen and with anti-IgG of chickens but did not react with
antibody to parasite antigens.

Since globulin associated serum antigen and its antibody were
implicated in both anemia and nephritis, further study was made. Serum
antigen from rats with acute Babesia rodhaini was used to immunize
chickens. The globulins from these chickens reacted in serologic tests
with the plasma of chickens with acute P. gallinaceum infection, with
the antigens found in their kidneys, and with the antigen extracted
from droppings of malarious chickens. Conjugates of the antibody reacted
with the glomeruli of malarious chickens, and those of chickens with
plasma induced nephritis, in the same manner as did antibody to serum
antigen from malarious chickens.- Antibody to serum antigen of chicken
origin reacted in serologic tests with serum antigen in the plasma of
rats with acute B. rodhaini infection. Thus it was further indicated
that serum antigen was not a part of P. gallinaceum parasites. It
appears to be an antigen that is in some way elaborated during acute red
blood cell infections with parasites of other genera, as well as those
of genus Plasmodfium.

This work has incriminated two factors, the cold-active hemagglu—
tinin, and complexes of serum antigen and antibody in the anemia of
acute malarial infections of chickens. The nephritis of acute malaria
of chickens appears to be primarily an immune complex disease involving

serum antigen.

PATHOGENESIS OF ACUTE AVIAN MALARIA

By

Jiya Li Soni

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1973

Dedicated
to
my wife Mrs. Gayatri Soni
and

my family

ii

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to my advisor, Dr.
Herbert W. Cox, for his guidance and encouragement throughout these
studies and during preparation of the thesis.

Suggestions received from members of my guidance committee, Dr.
Gordon C. Carter, Dr. Jeffery F. Williams, Dr. Vance L. Sanger, Dr.
David H. Bing, and Dr. H. Donald Newson, are thankfully acknowledged.
I am also thankful to Dr. Kenneth K. Keahey and Dr. Richard A. Patrick,
who served on my guidance committee, and for their review of the
manuscript in the absence of Drs. Sanger and Bing, respectively.

The companionship and moral support received from my fellow
students, Mr. A. J. Musoke, Mr. W. Leid, Mr. P. G. Engelkirk, Dr.

P. B. Conran, and the technical assistance and moral support received
from Mrs. Marie Harding and Dwight L. Baily will be remembered with
affection.

All the graphs of this thesis were drawn by Mrs. Lacy C. Cox,
which I consider a token of her cordial generosity.

This program of advanced studies in the United States of America
and in the Department of Microbiology and Public Health at Michigan
State University was made possible by a fellowship from the United
States Agency for International Development. I am personally thankful
to Mr. Tom A. McCowen and Shirley Lovenguth of Overseas Project,
University of Illinois; Dr. Irving A. Wyeth and Patricia K. Riley of
International Agricultural Projects, Michigan State University; and

iii

Mrs. Lula Mae Dennison of the United States Department of Agriculture,
International Training, Foreign Development Division, Washington, D.C.,
for their support and cooperation.

I am also thankful to authorities of the Jawaherlal Nehru Krishi
Vishwa Vidyalaya, Jabalpur, M.P., India, for giving me this opportunity
to pursue a higher education abroad. I particularly appreciate the
kindnesses and inspiration from Dr. R. L. Kausal, Dean, Faculty of
Veterinary Science and Animal msbandry.

I wish to acknowledge the support received from funds from Grant
No. AI-80508 from the National Institutes of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, Maryland, and from

the Michigan Agricultural Experiment Station.

iv

TABLE OF CONTENTS
Page
mTRODUCTION O O O O O O O O o o O O O O O O O O O O O O O O O O O 1

REVIEW OF L ITERATURE O O O O O O O O O O O O O O I O O O O O O I O 3

Hemosporidian Parasites . . . . . . . . . . . . . . . . . . 3
Plasmodium gaZZinaceum Brumpt 1935 . . . . . . . . . 3
Immunological Studies . . . . . . . . . . . . . . . . . . . 8
Avian Immunology . . . . . . . . . . . . . . . . . . 8

Parasite Antigens . . . . . . . . . . . . . . . . . . . . . 10

Serological Cross Reactions of Plasmodial Antigens . 12
Antigens in Plasma. . . . . . . . . . . . . . . . . . . . . 13
Origin of Serum Antigens. . . . . . . . . . . . . . . . . . 16
The Role of Serum Antigens in the Pathogenesis of Disease . l7
Immunizing Properties of Serum Antigens . . . . . . . . . . 17
Cold-Active Agglutinin for Trypsinized Erythrocytes . . . . l9
Immunological Mechanisms in Malarial Disease. . . . . . . . 20

AneIIlj-a in Malaria. O O O O O O O O O O O O O O O O O 20
Malarial Nephropathy . . . . . . . . . . . . . . . . 22

REFERENCES 0 O I o O O O O O o O O O o O O o O O 0 O O O O O O O O 28

ARTICLE 1 PATHOGENESIS OF ACUTE AVIAN MALARIA. I. IMMUNO-
LOGIC REACTIONS ASSOCIATED WITH ANEMIA, SPLENO—
MEGALY, AND NEPHRITIS OF ACUTE PLASMODIW GALLI—
NACE'W INFECTIONS OF CHICKENS. . . . . . . . . . . . 40

ARTICLE 2 PATHOGENESIS OF ACUTE AVIAN MALARIA. II. A
STUDY OF ANTIGENS AND ANTIBODIES ASSOCIATED WITH
ANEMIA OF ACUTE PLASMODIUM’GALLINACEUM INFECTION
OF CHICKENS. . . . . . . . . . . . . . . . . . . . . 67

ARTICLE 3

ARTICLE 4

Page

PATHOGENESIS OF ACUTE AVIAN MALARIA. III.
IMMUNOLOGIC MEDIATORS OF NEPHRITIS IN ACUTE
PLASMODIUM GALLINACEUM INFECTIONS OF CHICKENS. . . . 102

PATHOGENESIS OF ACUTE AVIAN MALARIA. IV.

ANEMIA MEDIATED BY THE COLD-ACTIVE AGGLUTININ

FOR TRYPSINIZED ERYTHROCYTES FROM THE BLOOD OF

CHICKENS WITH ACUTE PLASMODIUM GALLINACEUM

INFECTION. . . . . . . . . . . . . . . . . . . . . . 138

vi

LIST OF TABLES
Table Page

ARTICLE 1 PATHOGENESIS OF ACUTE AVIAN MALARIA. I. IMMU-
NOLOGIC REACTIONS ASSOCIATED WITH ANEMIA, SPLENO—
MEGALY, AND NEPHRITIS OF ACUTE PLASMODIUM
GALLINACEUM INFECTIONS OF CHICKENS

1 Average of percentage of parasitized erythrocytes (ZPE),
red blood cell counts (RBC x 106), spleen volume in ml.,
severity of kidney damage (SKD), the titers of hemag-
glutinin for trypsinized erythrocytes (HA), serum
antigen (SA) and antibody to serum antigen (ABSA) in
chickens infected witth8 PZasmodium gallinaceum infected
erythrocytes. . . . . . . . . . . . . . . . . . . . . . . .

2 Averages of percentage of parasitized erythrocytes (ZPE),
red blood cell counts (RBC x 106), spleen volume in ml.,
severity of kidney damage (SKD), the titers of hemag—
glutinin for trypsinized erythrocytes (HA), serum
antigen (SA) and antibody to serum antigen (ABSA) in
chickens infected with 106 Plasmodfium gallinaceum infected
erythrocytes. . . . . . . . . . . . . . . . . . . . . . . . 59

3 Average red blood cell counts (RBC x 106) on normal
chickens injected with the plasma of chickens with acute
Piasmodium gallinaceum infected (Exptl.) and normal
chickens injected with plasma of normal birds (Control) . . 60

4 Red blood cell counts (RBC x 106) of normal chickens
injected with "eluate" from P. gaZZinaceum infected
erythrocytes and normal chickens injected with "normal"
red cell eluate . . . . . . . . . . . . . . . . . . . . . . 61

5 Severity of kidney disease (SKD) in normal Chickens and
in chickens recovered from Plasmodium gallinaceum 24
hours after intravenous injection of plasma of chickens
with acute malaria. . . . . . . . . . . . . . . . . . . . . 62

ARTICLE 2 PATHOGENESIS OF ACUTE AVIAN MALARIA. II. A
STUDY OF ANTIGENS AND ANTIBODIES ASSOCIATED WITH
ANEMIA OF ACUTE PLASMODIUM GALLINACEUM INFECTION
0F CHICKENS

1 Red blood cell counts (RBC x 106) of normal chickens
after injection of plasma from chickens with acute Plas-
modium gallinaceum infection (Exptl.) and plasma of
normal chickens (Control) and the percent of RBC lost
(Z RBC lost). . . . . . . . . . . . . . . . . . . . . . . . 91

vii

Table Page

2 Titers of serum antigen (SA) and antibody to serum
antigen (ABSA) in plasma (I), saline washings of blood
cells (II), hypertonic saline leach of unwashed cells
(III), and leach from washed cells (IV) from the blood
of chickens sacrificed at 6, 12, 18 and 24 hours after
the injection of 4 ml of plasma from chickens with
acute Plasmodium gaZZinaceum malaria, and in chickens
injected with plasma of normal chickens.* Tested with
the tube bentonite flocculation test using bentonite
treated with SA from rats with acute babesiosis (R) and
from chickens with acute malaria (C) to detected ABSA,
and ABSA of both rat and chicken origin to detect SA. . . . 92

ARTICLE 3 PATHOGENESIS OF ACUTE AVIAN MALARIA. III.
IMMUNOLOGIC MEDIATORS OF NEPHRITIS IN ACUTE
PLASMODIUM GALLINACEUM INFECTIONS OF CHICKENS

l The average percent of parasitized erythrocytes (PE),
the red blood cell counts (RBC X 105), the average of
titers of serum antigen (SA) and antibody to serum
antigen (ABSA) in plasma, and fecal droppings from
chickens with acute PZasmodium gallinaceum malaria. . . . . 123

2 Parasitemia (av. Z PE) and anemia (av. RBC X 106) in
chickens infected with Plasmodium gallinaceum, the
titer of serum antigen (SA) and antibody to SA (ABSA)
in plasma, the intensity of fluorescent activity in
fluorescent antibody tests (FAT) with conjugated anti-
body to serum antigen from rats with acute Babesia
rodhaini infection (ABr), antibody to serum antigen
from malarious chickens (ASA), antibody to P. gaZZina-
ceum parasite antigen (APA), and anti-chicken 7S
globulin, in frozen kidney sections from a chicken
brought to autopsy at daily intervals after infection . . . 124

ARTICLE 4 PATHOGENESIS OF ACUTE AVIAN MALARIA. IV.
ANEMIA MEDIATED BY THE COLD-ACTIVE AGGLUTININ
FOR TRYPSINIZED ERYTHROCYTES FROM THE BLOOD OF
CHICKENS WITH ACUTE PLASMODIUM GALLINACEUM
INFECTION

1 Average titers of cold-active agglutinin for trypsinr
ized human type "0" erythrocytes in the plasma of 5
chickens with acute Plasmodium gaZZinaceum infection,
3 normal chickens and 4 hyperimmunized chickens, before
and after absorption with the trypsinized cells at 4
C, the titers of the agglutinin in saline used to
elute cells at 37 C, and tests of each material for
agglutinin activity after 2-mercaptoethanol treatment
(Z-ME). . . . . . . . . . . . . . . . . . . . . . . . . . . 153

viii

Table

Average red blood cell counts (RBC x 106) on 4 chickens
(Exptl.) injected with cold agglutinin absorbed from
plasma of chickens with acute Plasmodium gallinaceum
infection with trypsinized human type "0" erythrocytes,
and on 4 chickens injected with control materials from
normal chicken plasma (Control) . . . . . . . . . . . .

Average red blood cell counts (RBC x 106) on 4 chickens
injected with plasma of chickens with acute.P1asm0dium
gaZZinaceum from which cold-active agglutinin had been
absorbed with trypsinized human type "0" erythrocytes

(Exptl.), and on 4 chickens injected with normal plasma
treated with the trypsinized cells (Control). . . . . .

The titers of cold-active agglutinin (CA), serum anti-
gen (SA) and antibody to serum antigen (ABSA) in plasma
from chickens with acute Plasmodium gaZZinaceum infec-
tion, before and after absorption‘with trypsinized
human type "0" erythrocytes . . . . . . . . . . . . .

ix

Page

154

155

156

LIST OF FIGURES
Figure Page

ARTICLE 1 PATHOGENESIS OF ACUTE AVIAN MALARIA. I. IMMU-
NOLOGIC REACTIONS ASSOCIATED WITH ANEMIA, SPLENO—
MEGALY, AND NEPHRITIS OF ACUTE PLASMODIUM
GALLINACEUM INFECTIONS OF CHICKENS

l Nephritis in acute Plasmodfium gallinaceum infections
of ChiCkenS C C C C C C C C C C C C C C C C C C C C C C C C 64

2 Nephritis in chickens induced by injection of plasma
of chickens with acute PZasmodium gaZZinaceum infection . . 66

ARTICLE 2 PATHOGENESIS OF ACUTE AVIAN MALARIA. II. A
STUDY OF ANTIGENS AND ANTIBODIES ASSOCIATED WITH
ANEMIA OF ACUTE PLASMODIUM GALLINACEUM INFEC-
TION OF CHICKENS

l The serologic relationships of antigens found in the
blood of chickens with acute Plasmodium gallinaceum
infection, and the physicochemical properties of
material leached from cells of malarious blood. . . . . . . 94

2 Elution profile after column chromatography in Sephadex,
of plasma of malarious chickens, material leached from
cells of malarious chicken blood with hypertonic (1.2%)
NaCl solution, and extract from Plasmodium gallinaceum
parasites. The columns were 2.5 x 100 cm, the packed
column size 2.5 x 90 cm, and columns were charged with
160 mg protein. Elution was with borate buffered
saline, pH 8.2, I = 0.16, the flow rate was 15 ml per
hour, and the fraction volume 3 ml/tube. A11 columns
were run at 22 C. . . . . . . . . . . . . . . . . . . . . . 96

3 The relationships of parasitemia (average Z parasi-
tized erythrocytes), anemia (average RBC counts x
106), and average antibody titers against serum anti-
gen, leached antigen, and parasite antigen in the
blood of chickens throughout the course of PZasmodium
gallinaceum infection. Titers were determined with
the tube bentonite flocculation test. Serum antigen
from rats with acute Babesia rodhaini infection, as
well as serum antigen from malarious chicken plasma,
was used to test for antibody to serum antigen. . . . . . . 98

Figure Page

4 Reactions of the blood cells of normal chickens that
had been injected intravenously with the plasma of
chickens with acute PZasmodium gaZZinaceum infections,
with fluorescein isothiocyanate conjugates of antibody
to serum antigen of rats with acute Babesia rodhaini
infection (ABr) prepared in chickens, antibody to
serum antigen from malarious chickens prepared in
chickens (ASA), antibody to chicken 78 globulin (IgG)
(commercial, prepared in rabbit), and antibody to
P. gaZZinaceum parasite antigen (APA) prepared in
chickens. . . . . . . . . . . . . . . . . . . . . . . . . . 100

ARTICLE 3 PATHOGENESIS OF ACUTE AVIAN MALARIA. III.
IMMUNOLOGIC MEDIATORS OF NEPHRITIS IN ACUTE
EIASMODIUM GALLINACEMM INFECTIONS OF CHICKENS

1 Reactions of antigen extracted from the kidneys (KA)
of chickens with acute PZasmodium gallinaceum
infection C C C C C C C C C C C C C C C C C C C C C C C C C 127

2 Tests of antibody extracted from the kidneys (KAb)
of chickens with acute Plasmodium gallinaceum
infection . . . . . . . . . . . . . . . . . . . . . . . . . 129

3 Reactions of extracts of droppings from chickens
with PZasmodium gallinaceum malaria . . . . . . . . . . . . 131

4 Elution profile of protein eluted from extracts of
droppings from chickens with acute Plasmodfium gaZZina-
ceum malaria, after column chromatography with Sephadex
G-100. Column size: 2.5 x 100 cm. Packed column:
2.5 x 90 cm. Charge: 4 m1 of extract. Elution with:
Borate buffered saline, pH 8.2, I = 0.16. Flow rate:
15 m1/hour. Fraction volume: 3 m1 . . . . . . . . . . . . 133

5 Reactions of kidney sections from chickens with acute
Piasmodium gaZZinaceum malaria, with fluorescein iso-
thiocyanate conjugated antibody to serum antigen
from rats with acute Babesia rodhaini infection (A),
antibody to serum antigen from chickens with acute P.
gallinaceum malaria (B), antibody to P. gallinaceum
parasite antigen (C), and anti-chicken 7S globulin
(D) (400 X) . . . . . . . . . . . . . . . . . . . . . . . . 135

6 The reactions of kidney sections from chickens taken
48 hours after injection of plasma from chickens with
acute Piasmodium gaZZinaceum malaria, with fluorescein
isothiocyanate conjugated antibody to serum antigen
from rats with acute Bhbesia rodhaini infection (A),
antibody to serum antigen from chickens with acute P.
gaZZinaceum malaria (B), anti-chicken 7S globulin (C),
and antibody to P. gaZZinaceum parasite antigen (D)
(400 X) . . . . . . . . . . . . . . . . . . . . . . . . . . 137

xi

Figure

Page

ARTICLE 4 PATHOGENESIS OF ACUTE AVIAN MALARIA. IV.

ANEMIA MEDIATED BY THE COLD-ACTIVE AGGLUTININ
FOR TRYPSINIZED ERYTHROCYTES FROM THE BLOOD OF
CHICKENS WITH ACUTE PLASMODIUM GALLINACEUM
INFECTION

The titers of cold-active agglutinin for trypsinized

human type "0" erythrocytes in column fraction samples

after column chromatography with Sephadex G-200 of

globulin from chickens with acute Plasmodium gaZZina-

ceum infection. . . . . . . . . . . . . . . . . . . . . . . 158

The serologic and physicochemical properties of cold—

active agglutinin absorbed from the plasma of chickens

with acute PZasmodium gaZZinaceum malaria at 4 C with
trypsinized human type "0” erythrocytes and eluted

from the cells at 37 C. . . . . . . . . . . . . . . . . . . 160

xii

INTRODUCTION

Anemia and changes in the vascular system that were manifested as
glomerulonephritis have long been a recognized part of malarial disease.
[Earlier investigators had indicated that anemia had been a result of
disruption of the erythrocytes by emerging parasites and that the
'vascular changes associated with nephritis might have been caused by
such mechanisms as anoxia resulting from blood loss, toxic substances
of the parasite, or blockage of blood vessels by parasites released
from blood cells by hemolysis (Maegraith, 1948). More recent studies
have furnished sufficient evidence to warrant the presumption that
anemia and nephritis of acute malaria, and other red blood cell infec-
tions, were mediated by immune mechanisms. Various investigators have
furnished evidence allowing the suggestion that malarial anemia and
nephritis might have been mediated by auto-antibody (Zuckerman, 1960;
McGhee, 1960; 1964; Cox et al., 1966; Iturri and Cox, 1969). Others
have suggested that both anemia and nephritis of acute malaria were
mediated by antigen-antibody complexes, which in combination with
complement might act as a hemolysin, or as a vascular permeability
factor to produce nephritis (Dixon, 1966; 1972; Ward and Conran, 1966;
1969). These suggestions have been based in large part on the findings
of agglutinins for trypsinized erythrocytes, or a positive Coomb's test,
associated with malarial anemia, and on the detection of immune gamma
globulin (IgG) in nephrotic lesions of the kidney, with fluorescein

isothiocyanate conjugated anti-IgG.

2

The definitive experiments, which would in essence fulfill Koch's
postulates for the definition of the etiologic agent of an infectious
disease, have not been performed. Neither malarial anemia nor nephritis
had been induced in experimental animals in absence of infection by
injecting them.with the immune substances found associated with acute
malarial disease. Neither have the antigens, which would be the essenr
tial ingredient for immunological diseases, either autoimmune or immune
complex, been identified and their roles in disease indicated.

It is therefore believed that the evidence that immune mechanisms
are the mediators of malarial nephritis and anemia is at best presumptive.
There is a real need for definitive experiments that prove a causal
relationship of these immune activities in absence of infection.

In the research for this thesis, using as a model PZasmodEum
gallinaceum infections of White Leghorn cockerels, an effort has been
made to implicate immune activity associated with acute infection in
both anemia and nephritis, and to indicate the antigens responsible for
immunopathogenesis. The review of the background literature, the metho-

dology, and the results of this research are presented.

REVIEW OF LITERATURE

Hemosporidian Parasites

 

Taxonomically hemosporidian parasites belong in the Phylum Protozoa
and fall within the Class Sporozoa, Order Hemosporidia. The families
Of the Class which are important from a pathological point of view are
the Plasmodiidoe, Hemoproteidoe, Theileriidde, and Babesiidbe and the
species which have most significance as causes of disease in man and
domestic animals are members of the genera Plasmodium, Hemoproteus,
Leucocytozoon, Theileria, and Babesia (Garnham, 1966).

These parasites typically have an intraerythrocytic stage in the
definitive host and their life cycle is characterized by 2 or more
reproductive phases: sporogony, a sexual life cycle, which takes place
in the body of blood sucking arthropods, and schizogony, an asexual life

cycle, which occurs in erythrocytes of the vertebrate host.

PZasmodium gallinaceum Brumpt 1935

Discovery and natural transmission. P. gallinaceum was probably
seen for the first time by Brousais in 1910 at Nhatrang, Indo-China,
in the blood of a native fowl. However, it was Brumpt (1935) who first
described the parasite under the name of Plasmodium gaZZinaceum.

Omer et a1. (1962) and Niles et al. (1965) found Aedés aegypti and
Mansonia crassipes, respectively, to be the natural vectors of P. galli-
naceum. Experimental infections of mosquitoes of the genera Aedés,
Aermigeres, Culex, Culiseta and Mansonia have been demonstrated (Brumpt,

3

4
1949), but A. aegypti is preferred for laboratory transmission since 100%
of fed mosquitoes may become infected, they are easily reared in the
laboratory, and will readily feed on many avian species. P. gaZZinaceum
was readily adopted to domestic chickens and has since been a popular
model for the study of plasmodial infections in both vertebrate and

mosquito hosts.

Developmental aspects in the avian host. Coulston, Cantrell and

 

Huff (1945) discovered the exoerythrocytic schizogony of plasmodial para-
sites using P. gaZZinaceum. Multiplication took place first in the
macrophages of the skin after introduction of sporozoites. The products
of fission were called cryptozoites. Subsequent fission produces meta—
cryptozoic stages. A morphologically distinct stage called phanerozoites
then developed which was capable of widespread invasion of the cells of
the lymphoid-macrophage system, and also the endothelial cells lining
the sinusoids and capillaries of internal organs. After two generations,
the progeny were capable of invading erythrocytes.

Huff (1952) recorded the frequency of metacryptozoite invasion of
different organs in experimentally infected birds in the following
order: spleen, lungs, heart, kidney, liver, brain, intestine, thymus,
and testes. Bray (1957) reported similar findings but also observed
parasites in cells of the pancreas and bone marrow.

Developmental and morphologic studies have been made on stages of
P. gaZZinaceum growing under several artificial situations. McGhee
(1949) found that sporozoites of P. gaZZinaceum will develop in the
lymphoid-macrophage system of 12- to l6-day-old chick embryos with
cryptozoic and metacryptozoic stages similar in duration and morphology
to those observed in hatched birds. Sporozoites will infect tissue

cultures of embryonic spleen cells where development takes place in the

5
macrophages, and Dobin et al. (1949) found as many as 12 parasites in a
single cell.

Studies of the nucleus and cytoplasm of erythrocytic schizonts in
tissue culture have been reported by Huff et al. (1960). Three develop-
mental forms of cytoplasmic division were observed which ultimately led
to cytomere formation and release of merozoites. Little diminution in
virulence or of the ability of the parasite to invade the erythrocytes
was found after the exoerythrocytic stage of P. gallinaceum had been
maintained in tissue culture continuously for 4 years (Meyer and
MUSacchio, 1963).

A fairly synchronous 36 hour schizogony cycle has been shown to
occur in blood (Giovannola, 1938). At the height of infection an
intense parasitemia occurs, at which time a single erythrocyte may
contain 2 or more trophozoites. At this stage the blood may contain
many immature erythrocytes. The increase in parasitemia continues
until there are more parasites than corpuscles. The acute phase lasts
for about 9 days, when it is terminated by a crisis.

Barretto and deFretes (1945) studied the correlation between age
and weight, and the mortality rate in P. gallinaceum infected chickens.
They found a 100% mortality in young chicks weighing less than 250
grams; 87% in chicks of 300 to 350 grams; and 45% in those weighing
1000 grams. The disease generally followed a milder course in adult
birds. The proportion of gametocytes to asexual parasites was always
low (1:100 to 2:100), but usually higher in the more acute attacks of

young chicks.

Ultrastructural studies related to Plasmodium gaZZinaceum. Rudzinska
and Trager (1957) share major credit for their pioneer work concerning

the fine structure of erythrocytic stages of malarial parasites. The

6
literature on ultrastructure has since become voluminous and only a few
pertinent references are cited.

Various organellae including a large nucleus, mitochondria with
microtubular cristae, well developed endoplasmic reticula, food vacuoles
with osmophilic malarial pigment particles, and double plasma membranes
were originally demonstrated and described by Rudzinska and Trager
(1957). Since then various new structures have been detected such as
the "conoid" and a system of peripheral fibrils, microtubules and convo-
luted tubules (Garnham et al., 1960; Garnham, 1961; Garnham et al.,

1961; Garnham et al., 1963).

In the erythrocytic stages of PZasmodium Zophurae parasites the
Observation of random invaginations of the parasite led Rudzinska and
Trager (1965) to suggest that they fed by intracellular phagotrophy.
Ristic and Kreier (1964) observed a similar phenomenon in their studies
of ultrathin sections of erythrocytic stages of P. gaZZinaceum. However,
in both erythrocytic and exoerythrocytic stages, a specialized structure
or "cytostome" has also been described on the surface of P. gaZZinaceum
(Meyer and Musacchio, 1965; Aikawa, 1966; Aikawa et aZ., 1966). Further
studies have detected differences in the size of the cytostome and
consequently of food vacuoles in different species, but Aikawa et al.
(1966) have suggested that the process of feeding is the same in both
avian and primate malarial parasites.

Garnham et al. (1960) carried out electron microscopic studies on
exflagellating gametocytes and described 3 zones. The outermost envelope
represented degenerating erythrocytic material. Next was a dense region,
corresponding to the cytoplasm of the parasite which contained the golgi
apparatus, endoplasmic reticulum, mitochondria, pigment, and the develop-
ing gamete, and finally there was a centrally located nucleus undergoing

endomitotic division.

Cara.

7

Histochemical studies have been made on different species of malarial
parasites, but few on P. gallinaceum, and the organism remains poorly
characterized in this reSpect. An electron microscopic cytochemical
study of glucose-6-phosphate-dehydrogenase (G-6-PD) activity in erythro-
cytes of malarious mice, monkeys, and chickens reported by Theakston and
Fletcher (1971) suggested that mammalian species of parasites have a
predilection for erythrocytes possessing detectable amounts of G-6-PD
activity. In P. gaZZinaceum infected chicken erythrocytes the distinc-
tion was much less obvious. No G-6-PD activity was found in parasites;
however, only a small proportion of chicken erythrocytes showed activity.
The study suggested that G-6-PD utilized by parasites was of erythrocytic
origin.

Histochemical tests using both light and electron microscopy have
been made for the presence of 3 acid hydrolases in plasmodia: acid
phosphatase, B-glucuronidase and aryl sulphatase were all found to be
located in lysosomes of some parasites (Scorza et al., 1972). All Of
the enzymes were detected in normal erythrocytes of each of the hosts;

however, P. gallinaceum parasites showed only aryl suphatase activity.

Pathology of P. ggZZinaceum infection. Most of the pathological
aspects of natural P. gaZZinaceum infection have been reviewed by Lund
and Farr (1965) and by Garnham (1966). The parasite is said to exert
its pathogenic effects in 2 ways; firstly by invasion of blood cells
and secondly by the development of exoerythrocytic stages in the brain.
Crawford (1945) described the disease as running a very acute course
in which birds lie in a corner with their faces and combs congested,
then rapidly become pale, weak, and diarrheic, and die within 7 days.
Quinine treated birds survived longer but eventually died due to

posterior paralysis. Intense parasitemia and profound anemia with an

8
erythrocyte count below 1 million cells per cubic millimeter was fre-
quently found. The anemia was also attributed to invasion of erythro-
poietic stem cells. A variety of pathological changes occur in the
acute stage of the disease but marked degenerative changes were seen
particularly in the adrenal glands, kidney, heart, and spleen along with
centrilobular necrosis of the liver. Death in cases of avian malaria is
generally considered to be due to cerebral lesions, resulting from capil—
lary blockage by schizonts from lysed red cells.

Taliaferro and Taliaferro (1955) made a correlative histopatho-
logical study with special reference to the phagocytic response in
avian malaria. Splenomegaly with black coloration due to malarial
pigment developed with damage to the malpighian bodies of the spleen
and lymphoid tissues very evident in advancing cases. An extensive
hyperplasia of lymphoid tissues occurred during recovery which they
ascribed to transformation of many cells to the macrOphage form.

Two sporadic outbreaks in domestic fowl have been reported from
India (Rao et aZ., 1951; Das et aZ., 1952). Parasites of one outbreak
were studied experimentally and a detailed account given of the clinical
signs, lesions, and immunity.

Jungle fowl are relatively resistant, but outbreaks of disease may
occur in domestic or newly introduced breeds of poultry in endemic

areas (Levine, 1967).

Immunological Studies

 

Avian Immunology_

 

Infectious diseases in chickens are evidently affected by immuno-
logical events involving both humoral and cellular responses and some

comments on the immunological system of avians are therefore pertinent.

9
The use of birds in experimental immunology has gained popularity since
the recognition of the role of the lymphoid tissues of the bursa of
Fabricius in the immune response (Pierce et al., 1966; Thorbecke at aZ.,
1968; Forget et aZ., 1970; Good, 1972). Lymphoid cells proliferating
within the bursal follicles first synthesize IgM and afterwards IgG
producing cells appear. Many experiments have now shown that this
change in immunoglobulin synthesis can occur only within the bursa of
Fabricius. Toivanen et a1. (1972) found that bursal lymphocytes given
in sufficient number to inbred chickens will completely reconstitute
the morphological characteristics and functional capacities of bursec-
tomized chickens, or of chickens made agammaglobulinemic by cyclophos-
phamide treatment in the neonatal period. On the other hand, cellular
immune events mediating delayed hypersensitivity and graft rejection
have been shown to be dependent upon the regulatory effect of the thymus
lymphocyte system (Brown, 1969; Good, 1972).

Primary immune responses in chickens are associated with the pro-
duction of IgM, followed by IgG. Thus 6-day antiserum to bovine serum
albumin contained mainly IgM antibody and synthesis of IgG followed
later (Benedict, 1967). Chickens inoculated with a single dose of
hapten-conjugated chicken protein produce antihapten IgG and an anti—
body identified as IgA (Dreesman et al., 1965). Three antigenically
and physicochemically distinct immunoglobulins have therefore been
reported to be present in chicken serum. However, the nature of secre-
tory immunoglobulins in chickens has been a matter of dispute. Leslie
et al. (1971) described a major secretory immunoglobulin from chickens
with a sedimentation value of 7 Svedberg units (S) which differed from
IgG and designated it "IgY." The 10.8 S moiety isolated from seminal

fluid of chickens was described as a dimer of IgY with or without

ad «P; T, a f; . n 1 1
1‘11: 1. 1 1. L e u .

n .
A.
.
Cu
I
.
l
C

.51. 1‘ u sly. A1. .3

10
secretory component. More recently Bienstock at al. (1973) described a
different immunoglobulin which they regard as the chief secretory com—
ponent and showed that it was 11.9 to 16.2 S IgA. Other immunoglobulins,
particularly those mediating immediate hypersensitivity, have yet to be
characterized.

Participation of humoral and cellular reSponses in human malarial
infection has been discussed and reviewed and IgM, IgG, and IgA have
been shown to possess specific antibody activity; however, protective
immunity was ascribed to IgG only (Turner and Voller, 1966; Rowe et aZ.,
1968; Brown, 1969). SO far there is little information in relation to
the specific immunoglobulins in the immune response of the chicken to
P. gaZZinaceum. In the following review an attempt will be made to
provide a comparative account of the antigens and antibodies which par-
ticipate in immune reactions and their consequences both in terms of
detection and the pathogenesis of the disease which is seen in malarious

birds.

Parasite Antigens

 

The chemical composition of PZasmodEum parasites has been extensively
studied, but characterization of antigens has been a more recent effort.
Antigenic analysis has been undertaken employing various physicochemical
and immunological techniques.

A number of antigens are thought to be shared by different species.
Indirect evidence suggestion of antigen-sharing by various plasmodial
Species has been provided by cross reactions in fluorescent antibody
tests and by passive hemagglutination tests (Voller, 1964; Bray, 1965;
C3<>llinset al., 1966), but each species also has its species specific

auntigens (Zuckerman and Spira, 1965). Spira and Zuckerman (1966)

11
obtained 12 to 16 protein bands in disc electrophoresis of cell free
extracts of most of the simian and avian malaria parasites, and the
major components were shown to be shared by different species. However,
the antigenic nature of these bands was not indicated.

Some of the difficulties encountered in the analysis of parasite
antigens are derived from the problem of separation of the organism from
host blood cells. Leukocytes have been found to interfere with the
efficient extraction and, accordingly, Sherman and Hull (1960), Diggs
(1966), and Sodeman and Meuwissen (1966) found it necessary to remove
the buffy coat during repeated washings of infected cells. Spira and
Zuckerman (1966) eliminated leukocytes and thrombocytes from their
preparation by employing 3 to 6% dextran solution in which the parasi-
tized red cells sedimented rapidly. Leukocytes may also be removed by
passing whole parasitized blood through a column of packed filter paper
(Aikawa and Cook, 1972).

In order to improve the purity of parasite antigens, attempts have
been made to extract parasitic material from a population of almost pure
infected cells. Separation of parasitized and non-parasitized cells has
been achieved by using sucrose density gradients or density gradient
mixtures of di-N—butyl and methyl-phthalate solutions. Parasite bearing
cells were found in the low density area (Williamson and Cover, 1966;

F. E. C. Cox, 1970; Saunders, 1970; Miller and Chien, 1970).

Various methods have been used for releasing plasmodial parasites
from infected cells, including saponin lysis, disruption by French pres—
sure cells, enzymic digestion, and immune lysis (Bangham and Horne,
1962; D'Antonio at al., 1966; 1970; Bahr, 1966; Turner and McGregor,
1969; Killby and Silverman, 1969). These methods were subjected to

evaluation by ultrastructural study of the released parasites by Aikawa

1"
out
‘7

.

p

L

a
1

Q

N

C

‘

 
 

o «11. 6L ~.: .ru. .1. ~\. v .\ ~ .. . a .. . .1: :c ., a .K» N

.L A. i. O n1 A . r . RIP. I. fix;

12

and Cook (1972) and their results suggested that saponin release of
malarial parasites was highly satisfactory. Different methods for the
extraction of antigens from released parasites have been used. Sonic
disruption, freezing and thawing, the Hughes press, and homogenization
in tissue grinders followed by extraction in veronal acetate buffer,
have all been used with varying success (Diggs, 1966; Zuckerman, 1966;
Turner and McGregor, 1969).

In general extracts prepared in these ways from different Plasmodium
Sp. have been shown to be extremely complex mixtures of protein, lipo-
protein, carbohydrate, and lipids. Antigenic activity has been most
often associated with proteinaceous moieties (Zuckerman, 1966). Immuno-
logical analysis and fractionation of parasite constituents of Plasmodium
berghei were carried out by Chavin (1966) and 5 rivenol precipitated
proteins were found to be antigenic when tested against serum of rats

immunized with P. berghei infection.

Serological Cross Reactions of Plasmodial Antigens

 

The occurrence of cross reactions between antigens within the genus
Plasmodium has already been referred to (Voller, 1964; Bray, 1965;
Collins at al., 1966). Kielman et al. (l970a,b) were able to use P.
gallinaceum infected erythrocytes as antigen for the immunofluorescent
diagnosis of human malaria infection. No difference in antibody titers
was observed using P. gaZZinaceum, Plasmodfium falciparum, PZasmodEum
cyanomolgican. berghei as antigen for the indirect immunofluorescence
test. High titers were seen in positive cases whereas in normal serum
samples no titers greater than 1:10 were Observed.

Species of Plasmodium and Babesia have been shown to share antigenic

substances. Serological cross reactions have been reported with sera of

13
recovered animals and serum of animals acutely infected with Plasmodium
and Babesia (Cox et aZ., 1968). This observation has been confirmed and
the indirect fluorescent antibody test used to study the relationship
betweeen antigens of Plasmodium vinckei, Plasmodium chabaudi, and P.
berghei and to those of Babesia rodhaini and Babesia microti. Cross reac-
tivity of antigens of Plasmodium folciparum, and Plasmodium vivax'with
Babesia argentina (Cox and Turner, 1970) was also found (Ludford et al.,
1972; Kagan et al., 1972). Additionally, a considerable degree of pro-
tective cross immunity has been shown to occur between malaria parasites

and piroplasms in rats and mice (Cox and Milar, 1968; Cox, l972a,b).

Antigens in Plasma

 

A variety of immunologic activities is detectable in plasma during
malarial infections. Blood of both experimental and naturally infected
animals has been shown to contain circulating antigenic substances which
have been designated as "serum antigens" (SA) or serum soluble antigens
(SSA). SA have been reported in monkeys with PZasmodEum knowlesi infec-
tion (Eaton, 1939; Cox, 1966), in ducks with P. Zophurae (Torry and Kahn,
1949) and in chickens with P. gaZZinaoeum (Todorovic et aZ., 1968;

Smith at aZ., 1969; Lykins at aZ., 1971). In human malaria infection SA
have been reported by Turner at al. (1971), McGregor et a1. (1968) and
Wilson at al. (1969). SA were associated with P. berghei malaria by Cox
at al. (1968), Wilson and Voller (1970), and Seitz (1972).

SA have been reported in other hemosporidian infections, particu-
larly in babesiosis in cattle (Mahoney, 1967), dogs, horses and rats
(Sibinovic et al., 1967a,b; 1969; Ristic at aZ., 1971).

SA generally appear during the acute phase of malaria infection and

persistence is variable (Turner and McGregor, 1969; McGregor, 1972). In

14
P. gallinaceum infected chickens SA first appeared at the peak of para—
sitemia and persisted in detectable quantities during the declining
phase of parasitemia over about 2 weeks (Todorovic et al., 1968).

In treated human patients with P. falciparum, relapse or recurrence
of parasitemia is followed by reappearance of SA but the specificity of
the SA produced differs according to the time at which the sample is
taken. Reappearance within a month leads to production of SA identical
to those seen in the acute phase of early infection. However, SA of
different specificities appear during parasitemia after one month has
passed (McGregor et aZ., 1968; McGregor, 1972).

The influence of treatment on persistence of SA has been discussed
at length by McGregor (1972). He reported that such antigens tend to
disappear quickly in patients responding effectively to antimalarial
therapy. However, the higher the titer at the time of treatment the
longer SA persisted in the circulation. The intensity of parasitemia
and the onset of production of antibody to SA were also found to affect
persistence (McGregor, 1972).

A considerable amountququantitative and qualitative data has been
accumulated on the nature of SA and it appears that many different
substances are involved. In early work SA with different precipitation
characteristics were described. Thus a SA which precipitated at pH
3.2 was reportedly present in greater concentration and was more stable
than a second antigen which precipitated at pH 5.6 according to Torry
and Kahn (1946). Acid precipitation at a pH lower than 5.5 led to loss
of antigenic activity (Eaton, 1939). In more recent work the SA present
in most human malarial infections have been found to be associated with
the macroglobulin peak when subjected to Sephadex G-200 gel filtration

(Turner, 1967; Turner and McGregor, 1968; Smith et al., 1969; 1970) and

15

the molecular weights have been variously estimated within the broad
range from 300,000 to 900,000 (Turner, 1967; McGregor et al., 1968).
For example, on the basis of gel filtration and thin layer Sephadex
G—200 chromatography, Wilson et al. (1969) determined that the SA in
P. falciparum designated '8' (heat stable) had a molecular weight of
400,000.

The physicochemical characteristics of several different SA in P.
gaZZinaceum have been used to classify these antigens as follows:
SAl is a protein with a molecular weight of 500,000 to 1,000,000; SA2
is a lipoprotein with a molecular weight of 150,000 to 250,000; and SA3 is
a protein of less than 70,000 mw. Other criteria such as the diffusion
rate in agar gel, sodium sulphate precipitation, anion exchange chroma-
tography and dextran sulphate precipitation were used to separate and
characterize SA in P. gallinaceum infection in the chicken. SAl was
precipitable with 10% sodium sulphate, and eluted from DEAE in 0.02 M
and 0.1 M phosphate buffer fractions. SA2 was precipitated with
dextran sulphate only (Lykins et aZ., 1971).

Using sucrose density gradient centrifugation, two fractions A and
B have been obtained and characterized from SA found in globulin of
horses, dogs and rats with acute babesiosis. Fraction A.was located in
the 25% sucrose zone and had a sedimentation coefficient of 88. Fraction
B was found in the 40% sucrose zone and had an S value of 20 to 23.
Physicochemical studies revealed them to be very complex structures
containing peptides, lipids, phosphatides and polysaccharides, all of
which were reported to contribute to antigenicity (Sibinovic at aZ.,
1967a).

Heat susceptibility has also been used to classify serum antigens

into S (stable), L (labile) and R (resistant) antigens both in man and

16

in owl monkeys (Wilson et al., 1969; Wilson and Voller, 1970). In 50
serum antigen samples at least 18 different S antigens have been
reported. S antigens in P. falciparum infection differ in physico-
chemical characteristics from those which occur in Babesia infections
and those in P. gallinaceum infection in chickens (Wilson et aZ., 1969).

Serum antigens of malarial infections have been discussed by Smith
et al. (1972) and the relationships of SA classified, following the

different criteria listed above.

Origin of Serum Antigens

 

The origin of serum antigens is still a controversial issue. On
the basis of their observations on the appearance of SA in P. gaZZina-
ceum infection at the peak of parasitemia and their persistence even
during the decline of parasitemia, when many erythrocytes are destroyed,
Todorovic et al. (1968) suggested that the origin of some SA could be
the parasitized erythrocytes themselves. 0n the other hand, Wilson
et al. (1969) have hypothesized that SA are derived essentially from
the parasite and suggest that either antigenicity is changed or a con-
siderable degree of antigenic heterogeneity is maintained within the
plasmodial population, which could account for the many SA detected.
However, the possibility that their '8' antigen was elaborated by
infected erythrocytes was not ruled out (Wilson et aZ., 1969).

It has been suggested by Turner and McGregor (1969a) that the
alpha SA antigen is a soluble product of infected cells whereas the
beta SA may be associated with the parasite itself. However, SA have
also been referred to as exo-antigens (Weitz, 1960; Fife, 1971) and
they have also been suggested to be secretions of excretions of the

parasite (Fife, 1971). In view of these many conflicting opinions, it

17
is not possible at this time to offer a definitive statement of the origin

of these substances.

The Role of Serum Antigens in the Pathogenesis of Disease

At the present time it is not clear whether serum antigens and
their antibodies have an important role in the pathogenesis of hemospor-
idian disease. It is generally conceded that anemia cannot be completely
attributed to the destruction of erythrocytes by the parasites, and
there is evidence relating SA to this phenomenon. Serum antigens of
acute malaria and babesiosis produced anemia when injected into normal
animals (Cox, 1966; Sibinovic at al., 1969). Adsorption of SA on the
surface of red blood cells has also been reported by Weitz (1960),
Zuckerman (1964a) and Sibinovic et a2. (1969). In discussing the
immunopathological mechanism of anemia in malarial infection Dixon
(1966) suggested that antigen-antibody complexes coating receptors on
the surface of RBCs are indeed considered to occur and, in association
with complement fixation, lead to opsonization or outright lysis of
erythrocytes. Such a mechanism involving serum antigen could have a
role in anemia.

Soluble antigens were suspected of being associated with glomerular
disease through the deposition of soluble immune complexes (Dixon,
1966; Cohen et al., 1969). Presumptive evidence of immune complex
deposits in malarial nephritis was obtained from renal biopsy specimens
by immunofluorescent staining with anti-IgG (Ward and Conran, 1966;

1969; Soothill and Hendrickse, 1967; Allison et al., 1969).

Immunizing_Properties of Serum Antigens

 

Although the results have not been uniformly consistent there has

accumulated over the past several decades considerable evidence to

 

 

3.. e
r- url
W2 kt .
nu A MAM
I.‘ D in Y
r
b R u.” 11m .1»
I... h 7..”
E vs
.1. 2 u _.
.. . . 1..
0 av ‘ 14‘
fi 5 I
.. a . .
5‘. Al 1. i
. i ,e .
. v a . . v .
A at
.C
. a w. :-
r 3.-
.q . ~z\ . r
. c a a
a a . fix . 4
I §\~ -
~ g h: Rn cm

“ Ap‘fli vw—vt p! \v‘

5. up...

a) .

18
suggest that protection against challenge may result from immunization
with serum antigens. In his classical early work Eaton (1939) observed
that soluble antigen from the serum of monkeys heavily parasitized with
P. knowZesi when injected into normal monkeys could produce complement
fixing antibodies similar to those seen in malaria infection. No pro-
tection seemed to have been produced against P. knowlesi challenge.
However, ducklings were found to be immunized against challenge after
inoculation with plasma from ducklings acutely infected with P.
Zophurae and serum of rats acutely infected with P. rodhaini, and more
than half of the immunized ducks showed significantly lower parasitemia
than that which developed in control birds (Corwin and Cox, 1969).
Similarly, rats immunized with serum antigens from P. knowlesi infected
monkeys also became resistant to challenge, in this case with P.
berghei (Cox, 1966); and plasma obtained from chickens infected with
P. gaZZinaceum has been used successfully to immunize chickens and
protect them against homologous challenge (Todorovic et aZ., 1967).

The phenomenon has been shown to occur with both homologous and
heterologous hemosporidian infections, for example, plasma taken from
rats or dogs with acute babesiosis was effective in immunizing rats
against B. rodhaini and dogs against B. canis (Sibinovic et al., 1967a,b).
Immunization has been achieved using serum antigens of Plasmodium or
Babesia species and challenge of the animals with another species (Cox,
1966; Sibinovic et al., 1967a; Corwin and Cox, 1969). Additionally
immunization by infection.with Plasmodium and Babesia species confers
resistance to challenge with heterologous species and genera of para—
sites (Cox and Miler, 1968; F. E. G. Cox, 1968). Immunogenicity of
serum antigens has therefore been shown to lack both parasite and host

specificity (Corwin and Cox, 1969).

”v-H

gar

-A
a.

3t

l9
Cold-Active Agglutinin for Trypsinized Erythrocytes

A cold-active agglutinin was detected in the sera of persons suffer—
ing from black water fever (Oliver-Gonzalez, 1944). He suggested that
it might contribute to the development of the disease.

An association of cold agglutinin for trypsinized red cells and
anemia and erythrophagocytosis in P. berghei and B. rodhaini infections
in rats was reported by Cox et a1. (1966) and Schroeder et a1. (1966).
They suggested that the agglutinin might have been stimulated by the
"T" stroma antigen of Hubner-Thomson-Fredenrich which were exposed by
enzyme treatment as was suggested by Springer (1963). Morton and
Pickles (1947) suggested.that such agglutinin was incomplete antibody
to erythrocyte antigens. Iturri and Cox (1969) reported that an antera—
tion in kidney vascular endothelium was associated with this agglutinin
and suggested that it might have a causal relationship.

Some physicochemical characteristics of cold agglutinins in infec-
tions of P. berghei in rats and P. Zophurae infected chickens have been
reported by Kreier et al. (1966) and Barrett et a1. (1970). In both
instances hemagglutinins were found to be more active at 4 C, occurred
in the macroglobulin fraction of serum and were eluted from erythrocytes
at 37 C. The agglutinins were susceptible to reductive cleavage by 2-
mercaptoethanol. Kano at al. (1968) observed an increase in IgA levels
in the serum of malaria infected humans and suggested that this may
represent cold agglutinin. These authors all speculated that hemagglu-
tinins were produced as a consequence of excessive erythrocyte destruc-
tion or to exposure of normally hidden antigenic sites on these cells.
Further reference to the role of cold agglutinins is made below in the

discussion of immune mechanisms in anemia in malaria.

 

I
u;
-(

n 'w
meta

2

«
aqp
L..C

IE

 

n.

|
.,.
.
F‘s

an

'5-

f

a v
‘k'u‘ '3 H

a“

20

Immunological Mechanisms in Malarial Disease

Anemia in Malaria

 

There are several disease entities in which the extent of the
anemia is not related directly to the number of infective agents in the
circulation and immunologic mechanisms have been suspected to be
responsible (Zuckerman, 1964a; McGhee, 1964; Cox et al., 1966; Schroeder
et al., 1966). In recent years a separate classification of immuno-
logically mediated anemias has been developed in which autoimmune
mechanisms associated with incomplete warm and cold agglutinins have
been implicated (Davidson and Neelson, 1969).

In human malarial infections it has long been known that the
severity of anemia is not always correlated with the degree of parasi-
temia and this observation has been extended to experimental models
(Zuckerman, 1966). Several complex mechanisms hypothesized for the
production of anemia by immunologic means have been discussed and
reviewed by Brown (1969), including the liberation of antigenically
altered red cell components from infected red cells, the combination
of parasite products with the erythrocytes, and the production of
parasite antigens closely resembling host components. These may be
further compounded by the development of rheumatoid factor or immuno-
conglutinin (Houba and Allison, 1966). Dixon (1966) considered that
the most likely explanation of the anemia was that antigen and antibody
complexes, unrelated to the erythrocytes, could be adsorbed onto the
surface of normal red cells which might then be lysed by a complement
dependent system or phagocytosed. Evidence for these various hypotheses
is derived from many different observations. Enhanced phagocytosis of

non-infected cells has been observed by many workers and this phenomenon

21
is particularly marked at, or just following, the anemic crisis, but
may persist for some time after the bulk of the parasites have been
removed from the circulation (Zuckerman, 1964a; 1966; McGhee, 1965; Cox
at al., 1966).

Serum antigens circulating in the plasma have been found to be
associated with anemia (Cox, 1966; Todorovic et aZ., 1966a; McGregor
et al., 1968). Cold active agglutinins for trypsinized erythrocytes
have been demonstrated with various hemosporidian infections (Cox et
aZ., 1966; Schroeder at aZ., 1966; Kreier et aZ., 1966; Iturri and Cox,
1969; Barrett et aZ., 1970). Although the role of hemagglutinins is not
clear, when these antibodies are present they are better related to the
onset of anemia than is parasitemia. Further, it has been suggested
that these hemagglutinins react with autoantigens that are on the
surface of erythrocytes which are exposed during infection or by enzyme
treatment (Schroeder at al., 1965a).

In P. gallinaceum infection in the chicken, Gautam et a1. (1970)
showed that immune globulin becomes coated onto infected erythrocytes.
This immune globulin agglutinated trypsinized erythrocytes from unin-
fected animals, whereas non-trypsinized erythrocytes were not agglutin—
ated. It was suggested by these authors that the mechanism of erythro-
cyte destruction in malaria directly depends on the presence of parasites
or some of their products which are absorbed from the blood stream onto
erythrocytes. On the other hand, splenomegaly and anti-erythrocyte
antibodies have been shown by Swann and Kreier (1973) to be produced as
a result of erythrocyte destruction and were felt to be responsible for
removal of damaged erythrocytes rather than causal in the anemia itself

in P. gallinaceum infection.

22
Malarial Nephropathy

The association of nephritis with malaria is historically well
established and was reported in chronic quartan malaria during the 19th
(zentury (Atkinson, 1884; Thayer, 1899; Bignami, 1900). Nephritis as a
<:omplication of malarial infection has been consistently observed since
that time (James, 1910; 1912; Clarke, 1912; Deeks, 1916; Goldie, 1930;
(Siglioli, 1930; Jansco and D'Angel, 1931). Nephrotic lesions are
observed in many instances of infectious anemia in domestic animals
(Banks et aZ., 1972). Malarial nephritis has not been reported in
avian species.

Although advanced glomerular changes were associated primarily with
chronic or recurrent cases of Plasmodium malariae infection, Giglioli
(1932) on the basis of histopathological findings in 5 fatal cases of
malarial nephritis, suggested that renal lesions in acute P. thciparum
and occasionally P. malariae infection may spontaneously cure, but may
sometimes cause chronic or even fatal kidney disease. Later, Maegraith
and Findlay (1944) observed in their pathological studies that in the
malarious kidney the lumina of the tubules were frequently filled with
"casts" which appeared to range from desquamated epithelial cells and
red blood cells to reddish-brown granules, the composition of which was
reported to be uncertain. The casts were most abundant in the distal
convoluted tubules, the ascending loop of Henle and in the collecting
tubules. Spitz (1946) also reported hypercellularity and swelling of
the glomerular tuft and established that tubular changes were restricted
in occurrence to the disease entity known as malignant tertian malaria.
Kibuka—Musoke and Butt (1967) categorized several basic histological
groups in 77 cases of malarial nephrotic syndrome which they studied.

A majority of the cases (55) fell under the description of proliferative

1'.)
D 1-

O 9‘
’14 -

1:;-

l
M.

23
glomerulonephritis which was further subdivided into 5 groups consisting
caf diffuse, lobular, focal, chronic, and minimal or "no change" types.
'In a smaller population of cases "membranous glomerulonephritis" was
used to denote thickening of the basement membrane of glomeruli without
jproliferation of the glomerular tuft or capsule. Similar pathological
observations have been reported by Burger et a1. (1967) in renal biOpsy
findings in persons suffering from P. falciparum malaria. The glomeruli
in these cases were found to be hypercellular, avascular and usually
filled the capsular space. Adhesion of the glomerular tuft to Bowman's
capsule was frequent. In addition, scattered glomeruli exhibited
thickening and splitting of glomerular basement membranes when treated
with periodic acid-Schiff stain (Soothill and Hendrickse, 1967).

The relationship of clinical nephritis to the immunological events
in serum sickness was shown by von Pirquet (1911), and subsequent studies
have led to the suggestion that immunological renal injuries can be
produced by two distinct means: (1) antibody against antigen fixed in
the kidneys (e.g., against glomerular basement membrane), (2) circulating
antigen and antibody complexes (Weigle, 1961; Unanue and Dixon, 1967;
Dixon, 1968; Carpenter, 1970).

These two types of nephritis differ in the deposition pattern of
immune SUbstances in the kidneys. In the latter type of nephritis
antigens of streptococcal, viral, staphylococcal, malarial, and D-
penicillin have been implicated (Dixon, 1966; 1968; 1972; Koffler et
al., 1967; Lambert and Dixon, 1968; Haslett at aZ., 1968; Jaffe et al.,
1968; West at aZ., 1968; Stickler et al., 1968; Stollerman and Pearce,
1968; Ward and Kibuka-Musoke, 1969; Banks et aZ., 1972; Banks and

Hanson, 1972).

24

The ultrastructure of such immune deposits in renal glomeruli has
‘been reviewed by Churg and Grishman (1972). Deposits differing in
composition, location and ultrastructural characteristics have been
described. In malarial nephritis electron microscopic observations
showed fusion of foot processes of epithelial cells and thickening of
the basement membrane. There is generally an increase in the subendo-
thelial zone as well as occasional aggregation of electron dense and
cytoplasmic material within the basement membrane itself (Allison et
al., 1969; Houba at al., 1971). The presence of small lacunae scattered
throughout the basement membrane is considered diagnostic of quartan
malarial nephrotic syndrome by Hendrickse at al. (1972). On the basis
of their ultrastructural studies, Boonpucknavig at al. (1973) concluded
that glomerular lesions in P. berghei infection were also induced by
immune complexes.

Similar electron microscopic observations have been made in ultra-
structural studies of immune complex nephritis due to other causes in
dogs (Murray et al., 1971; Halliwell and Blakemore, 1972; Kurtz et aZ.,
1972), in cats (Slauson at al., 1971), in glomerulitis of horses (Banks
et al., 1972), and in experimental renal disease induced by DNA—anti-
DNA immune complexes in rabbits (Natali and Tan, 1972).

The fluorescent antibody technique has been extensively used to
demonstrate the deposition of immune complexes in nephritis. The spe-
cific pattern of these complexes in glomeruli is in the form of fine,
granular or lumpy discontinuous deposits along the glomerular capillary
walls (Dixon et aZ., 1958; Dixon, 1963; Kniker and Cochrane, 1965).
This appearance differs markedly from the linear deposits of nephro-
toxic antibodies along the capillaries in nephrotoxic antiserum nephritis

described by Unanue and Dixon (1967). In recent studies employing

25

.fluorescein conjugated anti-IgG, IgM and 81C, evidence has been obtained
:suggesting that immunopathologic mechanisms in human malaria are mediated
‘tw'formation and localization of malarial antigen—antibody complexes
(ward and Conran, 1966; 1969; Dixon, 1968; Allison et al., 1969; Ward
and Kibuka—Musoke, 1969; Adeniyi et aZ., 1972; Hendrickse et al., 1972)-
IgG and 81C cryoglobulins have been incriminated as etiological agents
in acute glomerulonephritis, on the basis of granular deposits demonstrable
by immunofluorescence (Grupe, 1968). The characteristic deposition
patterns seen in FA studies have also been used to confirm immune complex
glomerulonephritis due to causes other than malaria (Slauson 8t aZ.,
1971; Murray at al., 1971; Porter and Porter, 1971; Oldstone and Dixon,
1971; Halliwell and Blakemore, 1972; Kurtz et al., 1972; Banks 8t aZ.,
1972; Natalli and Tan, 1972).

The pathogenic mechanisms of nephritis in autologous and heterolo-
gous phases have been-described at length by Unanue and Dixon (1967),
and Dixon (1972). A latent period was observed between the time at which
antigen became demonstrable in the glomeruli and the appearance of histo-
logical changes. During this latent period a continuous immunological
interaction in the glomerular capillaries occurred in which host anti-
bodies and complement were associating and dissociating from the most
permanent of the "planted" antigens (Unanue and Dixon, 1967). Small
complexes, found when there was antigen excess, tended to remain in the
circulation, whereas larger complexes, found at equivalence or in anti-
body excess, were rapidly removed by phagocytes. Intermediate sized
complexes, which were found in moderate antigen excess, may remain soluble
but large enough to react with complement. These were considered more
likely to be trapped in the vessel wall where focal inflammation may be

consequently induced (Dixon, 1968).

.
'1

.10

.~;-_~

kt;

D

I..-

35'
‘1.

u,A
N.‘

26

Cochrane and Dixon (1968) considered the likely sequence of action
for the pathogenic activity of immune complex nephritis to be as
follows: (a) formation of an antigen-antibody complex in the circula-
tion; (b) platelet clumping by the complexes with release of vasoactive
amines, (c) increased vascular permeability, (d) trapping of large
soluble complexes in the basement membrane and (e) complement fixation
and polymorphonuclear leukocyte accumulation.

Proteolytic enzymes and basic protein complexes are released which
"chew up" the basement membrane according to Dixon (1972). However, the
intensity and severity of lesions may also be dependent upon the meta-
bolic state of the glomerulus, the phagocytic mesangial cells, and the
degree of activity of the clotting system (Carpenter, 1970).

In spite of the growing mass of evidence concerning the immuno-
logic basis of malarial nephritis, few peOple have attempted to elute
antigens from affected kidneys. Antibodies were successfully eluted
using 0.1 M citric acid pH 2.5 (Allison et al., 1969), citric acid or
glycine buffer of pH 2.5 (Houba at al., 1971), by l M propionic acid
(Gallru 1970), or 0.02 M citrate buffer (Banks at aZ., 1972).

In conclusion, there is sufficient indirect evidence to warrant
the presumption that anemia and nephritis of acute malaria are mediated
by immune mechanisms. However, the definitive experiments in which the
disease signs were mediated by immune substances in absenceof infection
have not been performed. Neither has a comprehensive study of the
antigens, which are the essential ingredients of immunologic disease,
been made. Until it has been shown that a specific antigen and its
antibody can mediate a lesion and that they are present in the lesion,
the evidence for immune mechanisms in malarial disease is seriously

deficient. The objectives of this research are to produce malarial

27
disease, anemia and nephritis, without infection, and to identify the

antigens and antibodies responsible.

REFERENCES

REFERENCES

Ademiiyi, A., Akingkugbe, O. 0., Andres, G. A., Bassily, S., Brunner, T.,
Edington, G. M., Faulk, W. P., Goodman, H. C., Hendrickse, R. G.,
Houba, V., Kibukamusoke, J. W., Milgrom, F., Ogunlesi, T. O.,
Oyediran, A. B. O. 0., Soothill, J. F., and Soyannwo, M. A. O.

1972. Immunopathology of nephritis in Africa. Wld. Hlth. Org.
Bull. 31:387-396.

Aikznva, M. 1966. The fine structure of the erythrocytic stages of

three avian malarial parasites, Plasmodium faZZex, P. Zophurae
and P. cathemerium. Am. J. Trop. Med. Hyg. 15:449-471.

Aikenwa, M., Hepler, P., Huff, C. G., and Sprinz, H. 1966. The feeding
mechanism of avian malarial parasites. J. Cell. Biol. 28:355-373.

Aikenwa, M., and Cook, R. T. 1972. Plasmodium: Electron microscopy of
antigen preparations. Exp. Parasitol. 31:67-74.

Allaison, A. C., Hendrickse, R. G., Edington, G. M., Houba,.V., DePetris,
S., and Adeniyi, A. 1969. Immune complexes in the nephrotic
syndrome of African children. Lancet 1:1232-1238.

‘Atkubnson, I. E. 1884. Bright's disease of malarial origin. Am. J.
Med. Sci. 88:149.

Baht, G. F. 1966. Quantitative cytochemical study of erythrocytic
stages of Plasmodium Zophurae and P. berghei. Milit. Med. 131
(Suppl):1064-1070.

B'ET‘Jngham, A. D., and Horne, R. W. 1962. Action of saponin on biological
cell membranes. Nature 196:952-955.

Banks, K. L., Henson, J. B., and McGuire, T. C. 1972. Immunologically
mediated glomerulitis of horses. I. Pathogenesis in persistent

infection by equine infectious anemia virus. Lab. Invest. 26:
701-707.

Bankmg, K. L., and Henson, J. L. 1972. Immunologically mediated glomeru-
litis of horses. II. Antiglomerular basement membrane antibody

and other mechanisms in spontaneous disease. Lab. Invest. 26:
708- 715.

Barrétto, M. P., and deFreitas, J. L. P. 1945. Malaria aviaria. VII.
Algunes observacoes sobre os caracteres da infectas de Gallus
gaZZus pelo Plasmodium gallinaceum Brumpt 1935. Revta. Clin.

S. Paulo. 17:73-82.

28

Bar

6
uihU

nil-

«we‘v-
.a‘fi

 

k

‘.

v.1

"v

1»-

‘4

t

29

Barrett, J. T., Rigney, M. M., and Breitenbach, R. P. 1970. Character-
istics of the hemagglutinin produced during P. Zophurae malaria
in chickens. Infection and Immunity 2:304-308.

Berger, M., Birch, L. M., and Conte, N. F. 1967. The nephrotic syndrome
secondary to acute glomerulonephritis during falciparum malaria.
Ann. Int. Med. 67:1163—1171.

Bienenstock, J., Perry, D. Y. E., Gauldie, J., and Underdown, B. J. 1973.
Chicken 7A: Physico-chemical and immuno-chemical characteristics.

Boonpucknavig, V., Boonpucknavig, S., and Bhamarapravati, N. 1973.
Plasmodium berghei infection in mice: an ultra structural study
of immune complex nephritis. Am. J. Path. 70:89-108.

Bray, R. S. 1957. Studies on the exoerythrocytic cycle of the genus
Plasmodium. H. K. Lewis and Co., London.

Bray, R. S. 1965. The Stein and Desowitz hemagglutination test for the
detection of antibodies to P. berghei and P. vinckei. Ann. Soc.
Belge. Med. Trop. 45:397-402.

Brown, I. N. 1969. Immunological aspects of malaria infection. Adv.
in Immunol. 11:267-319.

Brumpt, E. 1935. Paludisme aviaire: Plasmodium gaZZinaceum n.sp. de la
poule domestique. C.r. hebd. Seanc. Acad. Sci. Paris, 200:783—786.

Brumpt, E. 1949. Précis de Parasitologie, 6th edition. Masson et Cie,
Paris.

Carpenter, C. B. 1970. Immunologic aspects of renal disease. Ann. Rev.

Chavin, S. I. 1966. Studies on antigenic constituents of PZasmodEum
berghei. I. Immunologic analysis of the parasite constituents.
II. Fractionation of the parasite constituents. Milit. Med. 131
(Suppl):1124-1136.

Churg, J., and Grishman, E. 1972. Ultrastructure of immune deposits in
renal glomeruli. (Review). Ann. Int. Med. 76:479-486.

Clarke, J. T. 1912. Nephritis and quartan fever. J. Trop. Med. Hyg.
15:133-134.

Cochrane, C. G., and Dixon, F. J. 1968. The textbook of immuno-
pathology, 94. (Miescher, P. A. and Mfiller-Eberhard, H. J. EDs.
Grune and Stratton, N.Y.). 1968.

Cohen, S., Butcher, G. A., and Crandell, R. B. 1969. Action of malarial
antibody in vitro. Nature 223:368-371.

30

Collins, W. E., Jeffrey, G. M., and Skinner, J. C. 1964a. Fluorescent
antibody studies in human malaria. 1. Development of antibodies
to Plasmodium malariae. Am. J. Trop. Med. Hyg. 13:1-5.

Corwin, R. M., McGhee, R. B., and Sloan, B. L. 1965. Anemia in duck-
lings injected with malarious plasma. J. Parasitol. 51(Sup):36.

Corwin, R. M., and McGhee, R. B. 1966. Anemia in ducklings treated
with malarious plasma. Exp. Parasitol. 18:281-289.

Corwin, R. M., and Cox, H. W. 1969. The immunogenic activities of the
nonspecific serum antigens of acute hemosporidian infections.
Milit. Med. 134(Special):1258—1265.

Coulston, F., Contrell, W., and Huff, C. G. 1945. The distribution and
localization of sporozoites and preerythrocytic stages in infec-
tions with P. gallinaceum. J. Infect. Dis. 76:226-238.

Cox, F. E. G. 1968. Immunity to malaria after recovery from piro-
plasmosis in mice. Nature 219:646.

Cox, F. E. G., and Turner, S. A. 1970. Antigenic relationships between
the malaria parasites and piroplasms of mice as determined by
the fluorescent-antibody technique. Bull. Wld. Hlth. Org. 43:

Cox, F. E. G. 1970. Separation of parasites in sucrose gradients.
Nature 227:192.

Cox, F. E. G. l972a. Protective heterologous immunity between P.
atheruri and other plasmodium spp and Babesia spp in mice.
Parasitology 65:379-387.

Cox, F. E. G.‘ 1972b. Immunity to malaria and piroplasmosis in mice
following low level infections with Anthemosoma garnhami
(Piroplasmea:Dactylosomidae), Parasitology 65:389-398.

Cox, H. W. 1966. A factor associated with anemia and immunity in
Piasmodflum knowlesi infections. Milit. Med. 131(Supp1):1195-
1200.

Cox, H. W., Schroeder, W. F., andeistic, M. 1966. Hemagglutination
and erythrophagocytosis associated with the anemia of P. berghei
infection of rats. J. Protozool. 13:327-332.

Cox, H. W., and Miler, R. 1968. Cross-protection immunization by
Plasmodium and Babesia infections of rat and mice. Am. J. Trop.
Med. Hyg. 17:173-179.

Cox, H. W., Miler, R., and Patterson, S. 1968. Serologic cross-
reactions of serum antigens associated with acute Plasmodium
and Babesia infections. Am. J. Trop. Med. Hyg. 17:13-18.

Crawford, M. 1945. Plasmodium gaZZinaceum, a malaria parasite of the
domestic fowl. Vet. Rec. 57:395-396.

31

D'Antonio, L. E., von Doenhoff, A. E., and Fife, E. H. 1966. A new
method for isolation and fractionation of complement fixing

antigens from Plasmodium knowlesi. Proc. Soc. Exp. Biol. Med.
123:30-34.

DlAntonio, L. E., Spira, D. T., Fu, R. C., Dagnillo, D. M., and Silverman,
P. H. 1970. Malaria resistance: Artificial induction with a
partially purified plasmodial fraction. Science 168:1117-1118.

Das, J., Rao, S. B. V., and Ramnani, D. R. 1952. Studies on Piasmodium
gaZZinaceum. Indian Vet. J. 29:14-26.

Davidson, 1., and Neelson, D. A. 1969. Blood: Immunohematology, ch. 5:
247-250. Todd and Sanford Clinical diagnosis by laboratory..
method, Davidson and Henry (Ed), 14th edi. W. B. Saunders Co.,
Philadelphia.

Decks, W. E. 1916. Cited by Maegraith (1948).

Diggs, C. L. 1964. Comments on the antigenic analysis of Plasmodia.
Am. J. Trop. Med. Hyg. 13:217-218.

Diggs, C. L. 1966. Immuno diffusion studies of Plasmodium berghei:
Interactions of an extract of the erythrocytic forms with rabbit
antisera. Exp. Parasitol. 19:237-248.

Dixon, F. J., Vazquez, J. J., Weigle, W. 0., and Cochrane, C. G. 1958.
Pathogenesis of serum sickness. AMA Arch. Path. 65:18-28.

Dixon, F. J. 1963. The Harvey lectures series 21:58. Academic Press.

Dixon, F. J. 1966. Comments on immunopathology. Milit. Med. 131(Suppl):
1233-1234.

Dixon, F. J. 1968. The pathogenesis of glomerulonephritis. Am. J.
Med. 44: 493-49 8.

Dixon, F. J. 1972. Glomerulonephritis and immunopathology. In
IMmunobioZogy by R. A. Good and D. W. Fisher (ed.) Chapter 17:
167-173. Sinauer Associates, Inc., Publisher. Stamford, Conn.

Dubin, I. N., Laird, R. L., and Drinnon, V. P. 1949. The development
of sporozoites of Piasmodium gaZZinaceum into cryptozoites in
tissue culture. J. Natn. Malar. Soc. 8:175-180.

Eaton, M. D. 1939. The soluble malarial antigen in the serum of monkeys
infected.with Plasmodium knowlesi. J. Exp. Med. 69:517-532.

Fife, E. H. Jr. 1971. Advances in methodology for immunodiagnosis of
parasitic diseases. Exp. Parasitol. 30:132-163.

Forget, A., Potworowski, E. F., Richer, G., and Borduas, A. G. 1970.
Antigenic specificities of bursal and thymic lymphocytes in the
chicken. Immunology 19:465-468.

A I

a

G

‘1

znv

P.

U»

32

Gallo, G. R. 1970. Elution studies in kidneys with linear deposition
of immunoglobulin in glomeruli. Am. J. Path. 61:377-385.

Garnham, P. C. C., Bird, R.G., and Baker, J. R. 1960. Electron micro-
scopic studies of motile stages of malaria parasites. I. The
fine structure of the sporozoites of the Haemamoeba-(Plasmodium)
gallinaceum. Trans. Roy. Soc. Trop. Med. Hyg. 54:274-278.

Garnham, P. C. C., Bird, R. G., Baker, J. R., and Gray, R. S. 1961.
Electron microscopic studies of motile stages of malaria parasites.
II. The fine structure of the sporozoites of Laverani-(Plasmodium)
falciparum. Trans. Roy. Soc. Trop. Med. Hyg. 55:98-102.

Garnham, P. C. C., Bird, R. G., and Baker, J. R. 1963. Electron micro-
scopic studies of motile stages of malaria parasites. IV. The
fine structures of sporozoites of four species of Plasmodium.
Trans. Roy. Soc. Trop. Med. Hyg. 57:27-31.

Garnham, P. C. C. 1966. Malaria parasites and other haemosporidia.
Blackwell Scientific Publications, Oxford.

Gautam, O. P., Kreier, J. P., and Kreier, R. C. 1970. Antibody coating
on erythrocytes of chickens infected with Plasmodium gallinaceum.
Indian J. Med. Res. 58: 529- 543.

Giglioli, G. 1930. Malarial nephritis. J. and A. Churchill Limited,
London.

Giglioli, G. 1932. Clinical notes, autopsy and histopathological-find-
ings from five fatal cases of quartan malarial nephritis from
British Guiana. Trans. Roy. Soc. Trop. Med. Hyg. 26:177.

Giovannola, A. 1938. Piasmodium gallinaceum Brumpt 1935 i cosi ditti .
corpi Toxoplasma simili e1 alcune inclusioni di probabile natura
parassitaria ni globuli bianchi del Callus gaZZus. Riv. Parassit.
2:129-142.

Goldie, H. 1930. Notes on the association of malaria with nephritis.
Trans. Roy. Soc. Trop. Med. Hyg. 23:503.

Good, R. A. 1972. Structure-function relations in the lymphoid system,
pp 1-28, Chap. 1. Clinical Immunobiology, Vol. I, edited by
Bach and Good. Academic Press, New York.

Grupe, W. E. 1968. IgG-BlC cryoglobulins in acute glomerulonephritis.
Pediatrics 42:474-482.

Halliwell, R. E. W., and Blackmore, W. F. 1972. A case of immune-complex
glomerulonephritis in a dog. Vet. Rec. 90:275-280.

Hayslett, J. P., Bensch, K. G., Kashgarian, M., and Rosenbergh, L. E.
1968. Focal glomerulonephritis due to penicillamine. Lab.

 
 

..Q .nW.
.. .\ .1
4 m .. .a

I: . I. halfi

33

Hendrickse, R. G., Adeniyi, A., Edington, G. M., Glasgow, E. F., White,
R. H. R., and Houba, V. 1972. Quartan malarial nephrotic
syndrome. Collaborative clinicopathological studies in Nigerian
children. Lancet 1:1143-1149.

Houba, V., and Allison, A. C. 1966. M-antiglobulin (Rheumatoid-factor
like globulins) and other gamma globulins in relation to tropical
parasitic infections. Lancet 1:848—850.

Houba, V., Allison, A. C., Adeniyi, A., and Houba, J. E. 1971. Immuno-
globulin classes and complement in biopsies of Nigerian children
with the nephrotic syndrome. Clin. Exp. Immunol. 8:761-774.

Huff, C. G. 1952. Studies on the exoerythrocytic stage of Plasmodium
gaZZinaceum during the "transitional phase." Exp. Parasitol.
1:332-405.

Huff, C. G., Pipkin, A. C., Weathersby, A. B., and Jenssen, D. V. 1960.
The morphology and behaviour of living exoerythrocytic stages of
Plasmodium gaZZinaceum and P. fallex and their host cells. J.
Biophys. Biochem. Cytol. 7:95-102.

Iturri, G. M., and Cox, H. W. 1969. Glomerulonephritis associated with
acute hemosporidian infection. Milit. Med. 134(Special):1119-1128.

Jaffe, I. A., Treser, G., Suzuki, Y., and Ehrenreich, T. 1968.
Nephropathy induced by D-Penicillamine. Ann. Int. Med. 69:549-
556.

James, S. P. 1910. Quartan malaria and its parasites with special
reference to the occurrence of this form of malaria in the canal
zone. Cited by Maegraith, B. G. 1948. Pathological processes
in malaria and black water fever. Blackwell Scientific Publica-
tion, Oxford.

Kagan, I. G. 1972. Malaria: Seroepidemiology and serologic diagnosis.
Exp. Parasitol. 31:126-135.

Kano, K., McGregor, I. A., and Milgrom, F. 1968. Hemagglutinins in the
sera of Africans of Gambia. Proc. Soc. Exp. Biol. Med. 129:849-853.

Kibukamusoke, J. W., and Hutt, M. S. R. 1967. Histological features of
the nephrotic syndrome associated with quartan malaria. J. Clin.
Path. 20:117-123.

Kielmann, A., Weiss, N., and Sarasin, G.. 1970a. PZasmodEuM’gaZZinaceum
as antigen in the diagnosis of human malaria. Bull. Wld. Hlth.
Org. 43:612-616.

Kielmann, A., Sarasin, G., Bernhard, A., and Weiss, N. 1970b. ”Further-
investigation on Plasmodium gallinaceum as an antigen in the
diagnosis of human malaria. Bull. Wld. Hlth. Org. 43:617-622.

Killby, V. A. A., and Silverman, P. H. 1969.. Isolated erythrocytic
forms of P. berghei. An electron microscopical study. Am. J.
Trop. Med. Hyg. 18:836-859.

he

1'»..-

V
,‘v
~31

T
‘7

EVA
4“

I17:
I

‘C’

I)
t"

34

Kniker, W. T., and Cochrane, C. G. 1965. Pathogenic factors in vascular
lesions of experimental serum sickness. J. Exp. Med. 122:83-97.

Koffler, D., Schur, P. H., and Kunkel, H. G. 1967. Immunological
studies concerning the nephritis of systemic lupus erythematosus.
J. Exp. Med. 126:607-624.

Kreier, J. P., Shapiro, H., Dilley, D., Szilvassy, I. P., and Ristic, M.
1966. Autoimmune reactions in rats with P. berghei infection.
Exp. Parasitol. 19:155-162.

Kurtz, J. M., Russel, S. W., Lee, J. C., Slauson, D. 0., and Schechter,
R. D. 1972. Naturally occurring canine glomerulonephritis. Am.
J. Path. 67:471-482.

Lambert, P. H., and Dixon, F. J. 1968. Pathogenesis of the glomerulo-
nephritis of NZB/W mice. J. Exp. Med. 127:507-521.

Leslie, G. A., Wilson, H. R., and Clem, L. W. 1971. Studies on the
secretory immunologic system of fowl. I. Presence of immuno-
globulins in chicken secretions. J. Immunol. 106:1441-1446.

Levine, N. D. 1967. Protozoan parasites of domestic animals and of
man, 3rd printing, Burgess Publishing Co., 426 South Sixth Street,
Minneapolis, 15, Minnesota.

Ludford, C. G., Hall, W. T. K., Sulzer, A. J., and Wilson, M. 1972.
Babesia argentina, P. vivax, P. falciparum: antigenic cross
reactions. Exp. Parasitol. 32:317-326.

Lund, E. F., and Farr, M. M. 1965. Protozoa: Plasmodium infections.
pp. 1124-1132 in Diseases of’PbuZtry. Biester and Schwarte (Ed),
Fifth edition. The Iowa State Univ. Press, Ames, Iowa.

Lykins, J. D., Smith, A. R., Voss, E. W., and Ristic, M. 1971. Physical
separation of three soluble malarial antigens from the serum of
chickens infected with Plasmodium gallinaceum. Am. J. Trop. Med.
Hyg. 20:394-401.

Maegraith, B. G., and Findlay, G. M. 1944. Oliguria in black water
fever. Lancet 11:403-404.

Maegraith, B. G. 1948. Pathological processes in malaria and.blackwater
fever. Blackwell Scientific Publications, Oxford, England.

Mahoney, D. F. 1966. Circulating antigens in cattle infected with
Babesia bigemina or B. argentina. Nature 211:422.

McGhee, R. B. 1949a. Pre-erythrocytic development of Plasmodium
gallinaceum in avian embryos. J. Infect. Dis. 84:105-110.

McGhee, R. B. 1960. An autoimmune reaction produced in ducklings.in
response to injections of duck embryo blood infected with
Eflasmodfium Zophurae. J. Infect. Dis. 107:410-418.

35

McGhee, R. B. 1964. Autoimmunity in malaria. Am. J. Trop. Med. Hyg.
13:219-224.

McGhee, R. B. 1965. Erythrophagocytosis in ducklings injected with
malarious plasma. Prog. in Protozool. 11nd International Congress.
Protozool. London. P.171.

McGregor, I. A., Turner, M. W., Williams, K., and Hall, P. 1968.
Soluble antigens in the blood of African patients with severe
Plasmodium falciparum malaria. Lancet 1:881-884.

McGregor, I. A. 1972. Immunology of malarial infection and its possible
consequences. Brit. Med. Bull. 28:22-27.

Meyer, H., and Musacchio, M. D. E. O. 1963. Continuous maintenance of
Plasmodium gallinaceum in tissue culture. 7th Int. Congr. Trop.
Med. Malar. Rio de Janeiro.

Meyer, H., and Musacchio, M. D. E. O. 1965. An electron microscopic
study of the final and initial forms of Plasmodium gallinaceum
in thin sections of infected tissue culture. J. Protozool; 12:
193-202.

Miller, L. H., and Chien, S. 1971. Density distribution of red cells
infected by P. knowlesi and P. coatneyi. Exp. Parasitol. 29:

Morton, J. A., and Pickles, M. M. 1947. Use of trypsin in the detection
of incomplete anti-Rh antibodies. Nature 159:779-780.

Murray, M., Pirie, H. M., Thompson, H., Jarrett, W. F. H., and Wiseman,
A. 1971. Glomerulonephritis in a dog. A histological and
electron microscopical study. Res. Vet. Sci. 12:493-495.

Natali, P. G., and Tan, E. M. 1972. Experimental renal disease induced
by DNA anti DNA immune complexes. J. Clin. Invest. 51:345-355.

Niles, W. J., Fernando, M. A., and Dissanaike, A. S. 1965. Mansonia
crassipes as the natural vector of filaroidés, Plasmodium

gallinaceum, and other Plasmodia of fowls in Ceylon. Nature,
London, 205:411—412.

Oldstone, M. B. A., and Dixon, F. J. 1971. Immune complex disease in
chronic viral infections. J. Exp. Med. 134:32-40.

Oliver-Gonzalez, J. 1944. Blood agglutinins. Proc. Soc. Exp. Biol.
Med. 57:25-26.

Omar, A. R., Ismail, Y., and Lim, S. Y. 1962. Piasmodium gaZZinaceum
infections among chickens in Malaya. J. Malayan Vet. Med. Assoc.
3:75-80; 81-104.

Pierce, A. E., Chubb, R. C., and Long, P. L. 1966. The significance of
the bursa of Fabricius in relation to the synthesis of 7S and 19S
immune globulins and specific antibody activity in the fowl.
Immunology 10:321-337.

36

Porter, D. D., and Porter, H. G. 1971. Deposition of immune complexes
in the kidneys of mice infected with lactic dehydrogenase virus.
J. Immunol. 106:1264-1266.

Rao, S. B. V., Das, J., and Ramnani, D. R. 1951. Fowl malaria. Indian
Vet. J. 28:99.

Ristic, M., and Kreier, J. P. 1964. The fine structure of the erythro-
cytic form of Plasmodium gallinaceum as revealed by electron
microscopy. Am. J. Trop. Med. Hyg. 13:509-514.

Ristic, M., Lykins, J. D., Smith, A. R., Huxsoll, D. L., and Groves,
M. G. 1971. Babesia canis and B. gibsoni: soluble and corpuscu-
lar antigens isolated from blood of dogs. Exp. Parasitol. 30:
385-392.

Rowe, D. S., McGregor, I. A., Smith, S. J., Hall, P., and Williams, K.
1968. Plasma immunoglobulin concentrations in a West African
(Gambian) community and in a group of healthy British adults.
Clin. Exp. Immunol. 36:63-79.

Rudzinska, M. A., and Trager, W. 1957. Intracellular phagotrophy by
malarial parasites: an electron microscopic study of Plasmodium
Zophurae. J. Protozool. 4:190-199.

Rudzinska, M. A., Trager, W., and Bray, R. S. 1965. Pinocytic uptake
and the digestion of hemoglobin in the malaria parasites. J.
Protozool. 12:563-576.

Saunders, G. C. 1970. Concentration of plaque-forming cells by sucrose
density-gradient centrifugation. J. Reticulo-Endothelial Soc.
8:220-224.

Schroeder, W. F., Cox, H. W., and Ristic, M. 1965a. Erythrophagocytosis
and hemagglutinins associated with anemia and recovery from B.
rodhaini infections of rats. J. Parasitol. 51(Sec.2):30.

Schroeder, W. F., Cox, H. W., and Ristic, M. 1966. Anemia, parasitemia,
erythrophagocytosis and hemagglutinins in B. rodhaini infection.
Ann. Trop. Med. Parasitol. 60:31-38.

Scorza, J. V., Scorza, C., and Monteiro, M. C. C. 1972. Cyto-chemical
observations of three acid hydrolases in blood stages of malaria
parasites. Ann. Trop. Med. Parasitol. 66:167-172.

Seitz, H. M. 1972. Demonstration of malarial antigens in the sera of
Pflasmodium berghei infected mice. J. Parasitol. 52:179.

Sherman, I. W., and Hull, R. W. .1960. The pigment (hemozoin) and pro-

teins of the avian malaria parasite Plasmodium Zophurae. J.
Protozool. 7:409-416.

Sherman, I. W. 1964. Antigens of Plasmodium Zophurae. J. Protozool.
11:409-417.

fl.\U

37

Sibinovic, K. H., McLeod, R., Ristic, M., Sibinovic, S., and Cox, H. W.
1967a. A study of some of the physical, chemical and serologic
properties of antigens from sera of horses, dogs, and rats with
acute babesiosis. J. Parasitol. 53:919-923.

Sibinovic, K. H., Sibinovic, S., Ristic, M., and Cox, H. W. .1967b.
Immunogenic properties of babesial serum antigens. J. Parasitol.
53:1221—1229.

Sibinovic, K. H., Milar, R., Ristic, M., and Cox, H. W. 1969. In vivo
and in vitro effects of serum antigens of babesial infection and
their antibodies on parasitized and normal erythrocytes. Ann.
Trop. Med. and Parasitol. 63:327-336.

Slauson, D. 0., Russell, S. W., and Schechter, R. D. 1971. Naturally
occurring immune-complex glomerulonephritis in the cat. J. Path.
103:131-133.

Smith, A. R., Lykins, J. D., Voss, E. W., and Ristic, M. 1969. Identi-
fication of an antigen and specific antibody in the sera of
chickens infected with PLasmodium gallinaceum. J. Immunol. 103:
6-140

Smith, A. R., Karr, L. J., and Ristic, M. 1970. Characterization of
SA-l serum soluble antigen in chickens infected with PZasmodEum
gaZZinaceum. Fed. Proc. 29:637.

Smith, A. R., Karr, L. J., Lykins, J. D., and Ristic, M. 1972. Serum-
soluble antigens of malaria: a review. Exp. Parasitol. 31:120-
125.

Sodeman, W. A. Jr., and Meuwissen, J. H. E. T. 1966. Disc electrophore-
sis of Piasmodium berghei. J. Parasitol. 52:23-25.

Soothill, J. F., and Hendrickse, R. G. 1967. Some immunological studies
of the nephrotic syndrome of Nigerian children. Lancet 1:629-632.

Spira, D., and Zuckerman, A. 1966. Recent advances in the antigenic
analysis of Plasmodia. Milit. Med. 131(Suppl):lll7-1123.

Spitz, S. 1946. The pathology of acute falciparum malaria. Milit.
Surg. 99:555.

Springer, G. F. 1963. Enzymatic and nonenzymatic alterations of erythro-
cyte surface antigens. Bact. Rev. 27:191-227.

Stickler, G. B., Shin, M. H., Burke, E. C., Holley, K. E., Miller, R. H.,
and Segar, W. E. 1968. Diffuse glomerulonephritis associated
with infected ventriculoatrial shunt. New Engl. J. Med. 279:
1077-1082.

Stollerman, G. H., and Pearce, I. A. 1968. The changing epidemiology
of rheumatoid fever and acute glomerulonephritis. Adv. in
Internal Med. 14:201-239.

38

Swann, A. 1., and Kreier, J. P. 1973. Plasmodium gaZZinaceum: Mechan-
isms of anemia in infected chickens. Exp. Parasitol. 33:79-88.

Taliaferro, W. H., and Taliaferro, L. G. 1955. Reactions of the con-
nective tissue in chickens to Plasmodium gaZZinaceum and P.
Zophurae. I. J. Inf. Dis. 97:99-142.

Thayer, W. S. 1899. Cited by Maegraith, B. G. 1948.

Theakston, R. D. G., and Fletcher, K. A. 1971. An electron cytochemical
study of glucose-6-phosphate dehydrogenase activity in erythrocytes
of malaria-infected mice, monkeys and chickens. Life Sci. 10:

701-711.

Thorbecke, G. J., Warner, N. L., Hochwald, G. M., and Ohanian, S. H.
1968. Immunoglobulin production by the bursa of Fabricius of
young chickens. Immunology 15:123-134.

Todorovic, R., Ferris, D., and Ristic, M. 1967. Immunogenic properties
of serum antigens from chickens acutely infected with Piasmodium
gallinaceum. Ann. Trop. Med. and Parasitol. 61:117-124.

Todorovic, R., Ristic, M., and Ferris, D. 1968. Soluble antigens of
Piasmodium gaZZinaceum. Trans. Roy. Soc. Trop. Med. & Hyg. 62:
51-57.

Toivanen, P., Toivanen, A., and Good, R. A. 1972. Ontogeny of bursa
function in chickens. 1. Embryonic stem cell for humoral immunity.
J. Exp. Med. (in press). Cited by R. A. Good. 1972. Clinical
Immunogioloby, Vol. 1. Academic Press, New York.

Torrey, J. C., and Kahn, M. C. 1949. Precipitin test in Plasmodium
Zophurae malaria. J. Immunol. 62:41-48.

Turner, M. W., and Voller, A. 1966. Study on immunoglobulins of
Nigerians. I. The immunoglobulin levels of a Nigerian popula-
tion. J. Trop. Med. and Hyg. 69:99-103.

Turner, M. W. 1967. Fractionation of human malaria antigens and human
antimalaria antibodies in gel filtration. IProteides 0f the
Biological Fluids 15:591-596.

Turner, M. W., and McGregor, I. A. 1969a. Studies on the immunology
of human malaria. 1. Preliminary characterization of antigens in
fiasmodium falciparum infections. Clin. Exp. Immunol. 5:1-16.

Unanue, E. R., and Dixon, F. J. 1967. Experimental glomerulonephritis:
Immunological events and pathogenetic mechanisms. Adv. Immunol.
Vol. 6:1-90. Academic Press, New York.

Voller, A. 1964. Fluorescent antibody methods and their use in malaria
research. Bull. Wld. Hlth. Org. 30:343-354.

Ward, P. A., and Conran, P. B. 1966. Immunopathologic studies of simian
malaria. Milit. Med. 131(Suppl):1225-1232.

39

Warrl, P. A., and Conran, P. B. 1969. ImmunOpathology of renal compli-
cations in simian malaria and human quartan malaria. Milit.
Med. 134(Special):1228-1236.

Ward, 1P. A., and Kibukamusoke, J. W. 1969. Evidence for soluble immune
' complexes in the pathogenesis of the glomerulonephritis of quartan

malaria. Lancet 1:283-285.

Weigle, W. O. 1961. Fate and biological action of antigen—antibody
complexes. Advances in Immunology, Vol. 1:283-317.

Weitz, B. G. F. 1960. A soluble protective antigen of Trypanosoma
brucei. Nature 185:788-789.

West, C. D., McAdams, A. J., and Northway, J. D. 1968. Focalglomeru-
lonephritis in children. J. Pediat. 73:184-194.

Willsiaunson, J., and Cover, B. 1966. Separation of blood-cell-free
trypanosomes and malaria parasites on a sucrose gradient. Trans.

Roy. Soc. Trop. Med. Hyg. 60:425-427.

WilS<Jrl, R" J. M., McGregor, I. A., Hall, P., Bartholomew, R., and
Williams, K. 1969.. Antigens associated with Plasmodium falci-

parum infections in man. Lancet 2:201-205.

Wilson, R. J. M., and Voller, A. 1970. Malarial 'S' antigens from man
and owl monkey infected with Plasmodium falciparum. Parasitology,

61:461-464.

Z“‘3‘]l<‘-£.=..1:‘man, A. 1960. Autoantibody in rats with PLasmodium berghei.
Nature 185:189-190.

Zuckxetnnan, A. 1964a. Antigenic analysis of Plasmodia. Am. J. Trop.
Med. Hyg. 13:209-213.

zuckeIt‘man, A., and Spira, D. 1965. Immunoelectrophoretic comparison
of Plasmodial antigens. Wld. Hlth Org. Malaria Series 497/65:

1-6.

zuckeli‘tnan, A. 1966. Recent studies on factors involved in malarial
anemia. Milit. Med. 131(Suppl):1201-1216.

Article 1

PATHOGENESIS OF ACUTE AVIAN MALARIA
I. IMMUNOLOGIC REACTIONS ASSOCIATED WITH ANEMIA,
SPLENOMEGALY, AND NEPHRITIS OF ACUTE PLASMODIUM

GALLINACEUM INFECTIONS 0F CHICKENS

Jiya L. Soni and Herbert W. Cox
Department of Microbiology and Public Health,
Michigan State University

East Lansing, Michigan 48823

(To be submitted to Infection and Immunity)

PATHOGENESIS OF ACUTE AVIAN MALARIA
I. IMMUNOLOGIC REACTIONS ASSOCIATED WITH ANEMIA,
SPLENOMEGALY, AND NEPHRITIS OF ACUTE PLASMODIUM

GALLINACEUM INFECTIONS OF CHICKENS

Jiya L. Sonil and Herbert W. Cox

Department of Microbiology and Public Health,
Michigan State University,

East Lansing, Michigan 48823

'U}S. State Department, Agency for International Development,
Fe1143VV. Present address: College of Veterinary Science and Animal
H
“Shandry, Jawaharlal Nehru Agricultural University, Jabalpur, Madhaya

PradeSh , India.

40

 

41
ABSTRACT

Anemia, splenomegaly and glomerulonephritis in acute Plasmodium
gallinaceum infections of chickens were associated with cold-active
hemagglutinin for trypsinized human "0" erythrocytes, serum antigen, and
the concurrent presence of antibody to serum antigen, as well as with
the parasitemia of acute infection. Anemia with splenic enlargement
was produced in normal chickens within 24 hours by intravenous injection
of plasma from malarious chickens that contained high titers of hemag-
glutinin, serum antigen, and antibody. Similar anemia and splenic
engorgement resulted in normal birds from injection of eluates from the
washed cells of malarious chickens. The malarious plasma produced an
acute glomerulonephritis within 24 hours similar to that observed on
the 5th day of infection. The concurrent presence of the hemagglutinin,
serum antigen, and its antibody in the injected plasma, and the failure
of the plasma to produce nephritis in immunized chickens, suggested that

immune substances might be causal in acute malarial anemia, and nephritis.

ACKNOWLEDGEMENTS

Research here reported is from a thesis entitled, "Pathogenesis
of Acute Avian Malaria," submitted by the principal author, in partial
fulfillment of the requirements for the Doctor of Philosophy degree at
Michigan State University. Participation in this program was made
possible by a Fellowship from the U.S. State Department Agency for
International Development.

This research was supported in part with funds from Grant No.
AI-O8508 from the National Institute of Allergy and Infectious Disease,
National Institutes of Health, Bethesda, Maryland, and with support

from the Michigan Agricultural Experiment Station. This publication is

42
Journal Article No. 6469 from the Michigan Agricultural Experiment

Station.

INTRODUCTION

Anemia with splenomegaly, intravascular hemolysis and hemo-
globinuria are well known disease signs resulting from infection with
red blood cell parasites such as Plasmodium or Babesia species. Renal
complications have also been associated with acute malarial and babesial
infections, especially acute malignant tertian malaria and acute canine
babes iosis (16,17,26). In earlier reports blood loss was attributed to
red cell disruption by the emerging parasites, and mechanisms such as
anoxia following blood loss, parasite toxins, and blood-parasite thrombi
occlusion of the convoluted tubules have been suggested as causal in
the renal disease (16,17).

Anemia of acute malaria and babesiosis has been associated with
autoinlmune-like mechanisms. Acute blood loss and splenomegaly with
phago cytosis of uninfected erythrocytes were correlated with the presence
0f agglutinins for trypsinized erythrocytes (6,22). The induction of
anemia in normal animals by injection of globulin from animals with
acute malaria or babesiosis further indicated immune mechanisms in
blood loss. The injected globulins apparently became fixed to the
erYthrocytes causing them to be sequestered in the spleen (5,12,24).

The correlation of the severity of kidney damage with the titer
0f agglutinin for trypsinized erythrocytes prompted the suggestion that
autoimnlune mechanisms might be causal in the acute glomerulonephritis
ObserVed in acute Babesia rodhaiml infections of rats (13). However,
Ward and: Conran (30,31) suggested that antigens of the parasites were

the Substances principally responsible for stimulating the antibodies

involved. in nephritis of acute malaria.

43

Weigle (33) and Dixon (8) emphasized that soluble immune complexes
may cause increased vascular permeability, generalized anaphylaxis,
acute vasculitis and glomerulonephritis. It was also emphasized that
immune complexes could combine with red blood cells and act as opsonins
to cause phagocytosis or, in proper combination, fix complement and
cause hemolysis (10). Investigations of malarial nephritis indicated
that immune complexes might be present in kidney lesions (30,31,32).

In addition to cold-active hemagglutinin, serum antigens have
been detected in serum from animals with acute red blood cell infections
(3,5,7,23,25). Further, both serum antigen and its antibody may be
concurrently present in the blood of moribund animals (28). It seemed
possible that this antigen and its antibody could form complexes that
could combine with uninfected red cells causing them to be destroyed,
or could react with vascular endothelium to increase vascular
permeability.

We have undertaken a study that attempts to define the relation-
ship of the immunologic activities, found in blood during acute red
blood cell infection, to anemia and nephritis. The present communica-
tion reports results of experiments which indicated that immunologic
activity was related to anemia and nephritis in acute Plasmodfium

gallinaceum infections of chickens.

MATERIALS AND METHODS
P. gallinaceum was obtained from Dr. Julius P. Kreier, Faculty
of Microbiology and Cellular Biology, The Ohio State University,
Columbus, Ohio, and has been maintained by blood passage in White
Leghorn cockerels. The chickens were obtained from Rainbow Trails

Hatcheries, St. Louis, Michigan, as day-old chicks and were reared in

EDI

hen

net

be:

t
‘5
‘4‘:

"c
\-
v“\

44
a brooder to the age of 4 to 6 weeks prior to use. Inocula for passage
and for experiments were made with blood of infected roosters obtained
near the time of peak parasitemia in order to maintain maximum virulence
of the strain. Ten parts of blood, obtained from veins of the legs or
wings, were added to 1 part heparinized 0.78% NaCl solution (100 units
heparin per m1 saline). After centrifugation at 800 g for 10 minutes
the plasma was removed and the cells were resuspended in 0.78% saline.
Inocula were administered intravenously (IV) after determining the
number of parasitized erythrocytes (PE) per inoculum by described
methods (3).

Blood for Wright's stained smears or for RBC counts was obtained
by pricking the skin over the leg or foot veins. RBC counts were made
with Sahli dilution pipettes and a microscope hemacytometer. Blood for
experimental study was obtained by cardiac puncture under ether anes-
thesia, and was mixed with heparinized saline. Plasma was recovered
after centrifugation and stored at -18 C. Prior to use it was clari-
fied by centrifugation in a refrigerated centrifuge at 2000 g for 30
minutes.

Blood cells from infected chickens were washed twice with 0.78%
NaCl solution. A volume of 1.2% NaCl solution equal to the packed cell
volume was added with mixing and the mixture was allowed to stand over-
night at 4 C. The supernatant was recovered after centrifugation and
was dialyzed overnight at 4 C in 0.78% NaCl solution to reduce the salt
concentration. The volume of the supernatant was reduced to 1/10 of the
original by ultrafiltration in dialysis tubing under vacuum at 0 C. The
concentrated cell eluate was then dialyzed against 0.78% saline over-

night at 4 C and was then stored at -18 C until used.

7311:;
SET!

tio:

anes

line

for

plas

(7‘)

.
‘1.
§

L1‘
”as:
F!

‘ I
a
he

45

Serologic tests were performed with clarified plasma samples.
Titers of hemagglutinin (HA) for trypsinized erythrocytes were determined
by methods modified from those of Cox et al. (7), using trypsin-treated
human type "0" erythrocytes (1). Serum antigen (SA) and antibody to
serum antigen (ABSA) were assayed by means of a tube bentonite floccula-
tion (TBF) test described by Cox et a1. (7), and modified by Thoongsuwan
and Cox (28). To detect antigen, bentonite was treated with globulin
of rats recovered from babesiosis while globulin of rats with acute
Babesia rodhaini was used to measure antibody.

Chickens used for postmortem study were exsanguinated under ether
anesthesia and autopsied. The spleen was removed and its volume deter-
mined by water displacement. A kidney tissue sample 5 mm in thickness
was taken from the cortex area and was preserved in Bouin's fixative
for 6 hours. The samples were washed, processed and mounted in Para-
plast. Sections were cut at 5-6 u and stained with either Hematoxylin,
Giemsa's or Mallory's stain following recommended procedures (15).

The severity of kidney damage (SKD) was determined quantitatively
by microscopic study of 100 Nephrons (glomerulus and adjacent convo-
luted tubules) scoring severity from 0 to 4 and totaling the scores as
described (13).

Clarified plasma and the eluate from blood cells of malarious
chickens were tested for anemia inducing activity by IV injection into
normal birds using plasma and cell eluate from normal chickens as
controls. Blood counts were made before injection and daily thereafter
until the birds were discarded.

Normal chickens and those that had recovered from P. gaZZinaceum

infection were injected IV with malarious or normal plasma and 24 hours

46
later were exsanguinated and the kidney removed to be processed for

histopathologic study as described for malarious chickens.
EXPERIMENTAL RESULTS

Immunopathologic studies of acute avian malaria. Twenty-one
8-week-old cockerels were infected with 108 P. gallinaceum infected
erythrocytes. Three uninfected birds were autopsied and examined on day
zero and three of the infected birds were sacrificed at daily intervals
through the 5th day. Infections were patent on the day after inocula-
tion and the percentage of parasitized erythrocytes rose rapidly to 70%
by the 4th day. On the 5th day there was a drop in parasitemia which
was followed by death of all birds before the 6th day. The %PE, RBC
counts, score of kidney damage (SKD), spleen volume, and the titer of
SA, ABSA and HA are presented in Table 1.

A second experiment-was carried out using a smaller inoculum of
106 PE. Parasitemia was not detected in birds of this experiment until
the 3rd day and did not reach a peak until the 9th day. Thereafter,
there was a decline in parasitemia until the last of the birds were
sacrificed on the 16th day. Data on parasitemia red cell counts,
splenic enlargement, SKD, HA, SA and ABSA are presented in Table 2.

Gross observations of infected chickens showed that in birds
given the larger inoculum of parasites, cyanosis of the comb and
wattles was detected on the 3rd day and the birds had elevated tempera-
tures. During the 4th or 5th day they would suddenly develop subnormal
temperatures and became moribund. Combs and wattles would become
deeply cyanotic. A greenish diarrheal discharge was also evident.

The subnormal temperatures were followed by death within 24 hours.

Just prior to death, combs and wattles appeared blanched.

47

In birds infected with‘lO8 PE massive blood loss was evident on
the 5th day, but not until the 9th day in those infected with 106. Anemia
appeared to be better correlated with high titers of ABSA than to splenic
enlargement or the HA titer.

Nephropathic changes were detected early in both experiments but
scores were low. At this time, tests for both HA and SA were positive.
Marked increases in SKD values were associated with marked increases in
titers of ABSA. High SKD scores were indicated by swelling of the endo-
thelial cells of the capillary of the glomerulus which appeared to
obliterate the lumen of the capillary and cause the capillary loop to
completely fill Bowman's space. Increases in the number of mesangial
cell nuclei indicated a degree of hypercellularity of the tuft. There
was also extensive swelling of the epithelial cells of the adjacent
convoluted tubules that appeared to close the lumen, but without a
marked increase in the number of nuclei present. It was also noted
that blood vessels adjacent to the nephron were not patent and were
void of blood cells. A nephron appearing in this condition was given
an SKD value of 4. Study of the tubular elements reveals the presence
of hyaline casts and edema of the basement membrane (Figure l, A, B and

C).

Anemia from inoculations of plasma of malarious chickens. Normal
Chickens were injected IV with plasma of malarious chickens that had
been tested and found positive for HA, SA and ABSA. Prior to injection
red cell counts were made on each bird. The results of counts made on
normal birds injected with 2 m1 of malarious plasma and those given
normal plasma are shown (Table 3). The counts indicated that 24 hours

after injection the birds given malarious plasma had 31% fewer RBC than

48

birds given equal amounts of normal plasma. The counts were reduced
further to 41% two days after injection. Recovery from the blood loss
did not become apparent until after the 6th day.

Blood smears prepared from birds given the malarious plasma were
free of parasites on each day of observation. 0n the 11th day, 1 ml
samples of blood were taken from each chicken, pooled, and injected into
a normal chicken. Blood smears taken each day from this bird for 10

days showed no parasites.

Anemia from substances eluted from blood cells of malarious
chickens. Normal chickens inoculated IV with 2 ml of the eluate of
cells from malarious blood, and from cells of normal chickens, were
subjected to red blood cell counts daily (Table 4). The eluted super-
natant from normal chicken erythrocytes had no effect on red cell counts
of normal chickens while reduction in counts which persisted for 5 days
were observed in the birds injected with the solution eluted from erythro-

cytes of malarious birds.

_nghrit 3 induced with plasma of chickens with acute P. gaZZina-
gggm: A group of normal cockerels and another group that had recovered
from P. gallinaceum infection were each divided into two groups. One
group of normal and a group of the recovered birds were injected with
2 ml of plasma from malarious birds that contained HA, ABSA, and SA.
The other two groups were injected with the same amount of normal
chicken plasma. All of the birds were sacrificed 24 hours after
injection and kidney samples were taken for histologic study. The
SKD values estimated for each kidney are recorded (Table 5). The SKD
estimates for the chickens injected with the plasma of malarious birds

were as severe 24 hours after injection (average of 304) as was

49
observed on the 5th day (average of 274) in chickens infected with 108
P. gallinaceum infected erythrocytes, or on the 9th day (average 300)
in birds inoculated with 106 infected cells (Tables 1 and 2).
Histopathologic changes seen in kidneys of chickens 24 hours
after injection of normal plasma or plasma from malarious chickens are
shown (Figure 2, A, B, C and D). The nephron seen in Figure 2A shows
cellular hydration of endothelial cells of the glomerular tuft and of
the epithelial cells of the adjacent convoluted tubules similar to that
shown in Figure 1B. The presence of casts and changes in tubular
elements were noted (Figure 20). Cellular hydration of tuft endo-
thelium and tubular epithelium was not evident after injection of

malarious plasma into chickens that had recovered from acute P. galli—

naceum infection (Figure 2D).

DISCUSSION

It has been-shown that along-with severe anemia, there is an
acute glomerulonephritis associated with acute P. gaZZinaceum infections
of chickens. Nephritis became evident soon after the development of
patent parasitemia and increased markedly in severity with the appearance
of hemagglutinin for trypsinized erythrocytes, and antibody to serum
antigen in blood of the malarious birds. The sudden increase in
severity also preceded the anemia crisis and the profoundly moribund
condition usually noted just prior to the death of the birds.

The most severe form of the nephritis was manifested as cellular
hydration, or swelling of the endothelial cells of the glomerular tuft.
Capillary endothelium was swollen to the extent that the lumen of the
capillary was obliterated and the tuft completely filled Bowman's

capsule. The absence of blood cells in the capillary and in blood

50
vessels adjacent to the glomerulus suggested that blood supply to the
kidney might have been limited. Swelling of the epithelial cells of
the proximal convoluted tubules was also extensive and it appeared that
the lumen of the tubules were closed. The extent to which the blood
vessels and tubules appeared to be shut down and the number of nephrons
involved suggested that the kidneys of the birds might have been non-
functional during that stage of the disease. Evidence of extensive
extravasation of serous substance was indicated by the number of
hyaline casts seen in the lumen of the distal tubules. The histologic
appearance and the rapid onset of the changes are reminiscent of rapid
Changes observed in hydrocarbon-induced "toxic" nephritis described by
Smith and Jones (27).

In birds that survived acute malaria the changes observed in
moribund chickens were not evident. Nephropathic changes in these
birds would be best described as subacute or chronic as indicated by
some semilunar crescenting and fibrinous adherence of the tuft to
Bowman's membrane. While the glomerular side of the nephron later
appeared nearly normal, desquamated epithelium in the tubules remained
evident for some time.

This "toxic" nephritis syndrome was observed in normal chickens
24 hours after intravenous injection of plasma from malarious birds,
b1": was not produced in chickens that had recovered from acute P.
gallinacewn infection. The absence of plasma induced nephritis in
immune birds led us to suspect that immune mechanisms were responsible
for malarial nephritis.

The nephritis seen in malarious birds was similar in appearance
to that reported for rats with'acute B. rodhaini infection. Since, in

t
he rats, severity was correlated with the titers of agglutinin for

51

trypsinized erythrocytes, it was suggested that autoantibodies might
have been involved, and that the nephritis might have been similar to
that observed by Masugi (18) which was induced with antiserum to kidney
tissues (13). However, it is now known that serum of rats with severe
babesiosis may contain antibody to serum antigen and serum antigen as
well as the hemagglutinin (28). Thus, the nephritis of acute babesiosis
could have resulted from immune complexes of serum antigen and antibody.

Blood loss within 24 hours following the injection of plasma
from malarious chickens indicated the presence of anemia inducing
substances in the plasma. The observation of blood loss following the
injection of substances eluted from blood cells of malarious birds
suggested that the anemia factor(s) in plasma had combined with
erythrocytes.

Blood loss following injection of plasma of malarious animals
in these experiments confirmed earlier observations. Cox (5) reported
a sustained reduction in red cell counts of rats that had been inocu-
lated with globulin of monkeys with acute P. knowlesi malaria. Corwin
8t al. (4) observed blood loss in ducklings injected with plasma of
malarious ducks. Serum globulin of both rats and dogs with acute
babesiosis caused blood loss in normal rats following injection. That
globulin had combined" with erythrocytes was indicated by reactions of
the blood cells of the recipient rats with fluorescent antibody to
serum antigen. Since the reacting cells disappeared from the blood
and the spleens of the rats sacrificed for autopsy 24 hours after
inj e(:tion were enlarged, it was suggested that the cells had been
sequestered in this organ. The presence of large numbers of hyper-
c:hromic macrocytic erythrocytes in the blood of recipient rats 48

hours after injection indicated that the sequestered cells had been

d.-

0"?

.‘l.’

>r—1

52
destroyed. Since this phenomenon was not evident after injection of
normal globulin, blood loss was attributed to substances that had been
elaborated during acute babesial infection (24). In this earlier work
the only substance identified in the injected globulin was serum antigen.
We now know that serum of rats with acute B. rodhaini infection may
contain antibody to serum antigen as well, and it is now suspected that
Sibinovic et al. (24) may have been staining the antigen side of a
complex with the conjugated antibody.

It is possible that both anemia and acute nephritis might have
been in part mediated by the same immune substances. Since both serum
antigen and antibody were detected concurrently in plasma of moribund
birds it is possible that these ingredients could have been causal.

As was suggested by Dixon (10), we suspect that concentrations of
antigen and antibody might be obtained that would be optimal for bind-
ing erythrocytes, causing them to be sequestered in the spleen, or for
binding complement to cause intravascular hemolysis. As suggested by
Ward (29) complexes of antigen, antibody and complement may alter
vascular permeability, and thus in part contribute to nephritis. The
failure of malarious plasma to induce nephritis in immune recovered
birds encourages us to favor the idea that immune complexes, rather
than blood permeability factors, might have been causal. However,
permeability factors have been demonstrated in blood of malarious
animals (19,20,21). These too must be given due consideration in
determining the pathogenic mechanisms of vascular-renal disease associ-
ated with acute malaria.

We do not wish to imply that the observed nephritis was a major
cause of mortality. While it did appear that the kidneys were non-

furlCtional, this condition apparently existed for only a short time

53

and histopathologic study of kidneys of recovered birds did not reveal
that major damage had been done. It is probable that the capillary
beds of all major organs of the body were affected in a similar manner
and that malfunction of the brain or the lungs could have contributed
to death.

Finally, we would not wish to incriminate serum antigen and anti-
body as solely responsible for-morbidity and mortality in acute malaria.
In addition to the parasites, cold-active hemagglutinin was present,
and it is possible that there are other soluble antigens present in the
blood of malarious animals that might stimulate antibodies. Ultimate
definition of the causal substances depends upon isolation of each
immunologically active substance, purification of antigens, the detec-
tion of early appearing antibody and detecting the antigen and anti-
body in extravascular lesions with fluorescein conjugated monospecific

antisera. Attempts to achieve these definitive studies are in progress.

REFERENCES

l. Barrett, J. T., M. M. Rigney, and R. P. Breitenbach. 1970. Char-
acteristics of the hemagglutinin produced during Plasmodium
Zophurae malaria in chickens. Infec. Immun. 2:304-308.

2. Cooper, N. R., and B. J. Fogel. 1966. Complement in acute experi-
mental malaria. II. Alteration in the components of complement.
Mil. Med. (Suppl.) 131:1180-1190.

3. Corwin, R. M., and H. W. Cox. 1969. The immunogenic activities
of the nonspecific serum antigens of acute hemosporidian infec-

tions. Mil. Med. (Special Issue) 134:1258—1265.

 

4.

10.

11.

1:2.

1:3.

14.

54

Corwin, R. M., R. B. McGhee, and B. L. Sloan. 1965. Anemia in
ducklings injected with malarious plasma. J. Parasitol. (Suppl.)
51:36.

(30x, H. W. 1966. A factor associated with anemia and immunity in
.PZasmodium knowlesi infections. Mil. Med. (Suppl.) 131:1195-1200.

(30x, H. W., W. F. Schroeder, and M. Ristic. 1966. Hemagglutina-
tion and erythrophagocytosis associated with the anemia of

Eflasmodium berghei infections of rats. J. Protozool. 13:327-332.

' (Lox, H. W., R. Milar, and 8. Patterson. 1968. Serologic cross-

‘reactions of serum antigens associated with acute Plasmodium and
.Babesia infections. Amer. J. Trop. Med. Hyg. 17:13-18.

I)ixon, F. J. 1962. The Harvey Lectures. 21:58. Acad. Press.

I)ixon, F. J. 1965. Antigen-antibody complexes and autoimmunity.
.Ann. N. Y. Acad. Sci. lZ4(I):162-166.

I)ixon, F. J. 1966. Comments on immunOpathology. Mil. Med.
(Suppl.) 131:1233-1234.

]?ogel, B. J., A. E. von Doenhoff, Jr., N. R. Cooper, and E. H.
Fife, Jr. 1966. Complement in acute experimental malaria. I.
Total hemolytic activity. Mil. Med. (Suppl.) 131:1173-1179.

(Sautam, O. P., J. P. Kreier, and R. C. Kreier. 1970. Antibody
coating on erythrocytes of chickens infected with Plasmodium
gallinaceum. Indian J. Med. Res. 58:529-543.

IIturri, G., and H. W. Cox. 1969. Glomerulonephritis associated
‘with acute hemosporidian infection. Mil. Med. (Special Issue)
134:1119-1128.

BQibuka—Musoke, J. W., and M.S.R. Hutt. 1967. Histological fea-

tures of the nephrotic syndrome associated with quartan malaria.

J. Clin. Pathol. 20:117-123.

15.

16.

17.

18.

19.

20.

21-

22.

23.

 

55
Luna, L. G. 1968. Manual of histological staining methods of the

Armed Forces Institute of Pathology. Third Edition. McGraw Hill

Book Co. , N.Y.

Maegraith, B. C. 1948. Pathological processes in malaria and

black water fever. Blackwell Scientific Publications, Oxford,

England.
Malherbe, W. D., and B. S.Parkin. 1951. A typical symptomatology

in Babesia camls infection. J. S. African Vet. Med. Assoc. 22:

25-36.

Masugi, M. 1933. Beitr. Pathology Anat. U. Allgim. Pathol. 91:82.

Onbanjo, A. 0., A. R. Bhabani, and B. G. Maegraith. 1970. The
significance of kinin-destroying enzyme activities in Plasmodium
knowlesi malarial infection. Brit. J. Exp. Pathol. 51:534-540.

Onbanjo, A. 0., and B. G. Maegraith. 1970a. Kallikrein as a
pathogenic agent in Plasmodiwn knowlesi infection in Macaca
mulatta. Brit. J. Exp. Pathol. 51:523-533.

Onbanjo, A. 0., and B. G. Maegraith. 1970b. The probable patho-

genic role of adenosine in malaria. Brit. J. Exp. Pathol. 51:

581-586.
Schroeder, W. F., H. W. Cox, and M. Ristic. 1966. Anemia, para-

sitemia, erythrophagocytosis and'hemagglutinins in Babesia
rodhaini infections. Ann. Trop.'Med. Parasitol. 60:31-38.
Sibinovic, K. H., R. MacLeod, M. Ristic, S. Sibinovic, and H. W.
Cox. 1967a. A study- of some of the physical, chemical and sero-
logic properties of antigens from sera of horses, dogs and rats

with acute babesiosis. J. Parasitol. 53:919-923.

24.

.25.

:265.

22€3..

22$?..

30.

3]..

32.

56

Sibinovic, K. H., R. Milar, M. Ristic, and H. W. Cox. 1969. In
vivo and in vitro studies of the effects of serum antigens of
acute babesiosis and their antibodies on parasitized and normal
erythrocytes. Ann. Trop. Med. Parasitol. 63:327-336.

Sibinovic, K. H., S. Sibinovic, M. Ristic, and H. W. Cox. 1967b.
Immunogenic properties of babesial serum antigens. J. Parasitol.
53:1121-1129.

Siebold, H. R., and W. S. Bailey. 1957. Babesiosis (Piroplasmosis)
in dogs. J. Amer. Vet. Med. Assn. 130:46-48.

Smith, H. A., and T. C. Jones. 1966. Veterinary Pathology, Third
Edition, Lea & Febiger, Philadelphia, Pa.

Thoongsuwan, S., and H. W. Cox. 1973. Antigenic variants of the
hemosporidian parasite, Babesia rodhaini, selected by in vitro
treatment with immune globulin. Ann. Trop. Med. Parasitol.

(In press).

‘Ward, P. A. 1971. The role of complement in inflammation and
hypersensitivity. .13 H. Z. Movat (ed.) Inflammation, immunity
and hypersensitivity. Harper and Row, N.Y.

Ward, P. A., and P. B. Conran. 1966. Immunopathologic studies of
simian malaria. Mil. Med. (Suppl.) 131:1225-1232.

IJard, P. A., and P. B. Conran. 1969. Immunopathology of renal
complications in simian malaria and human quartan malaria. Mil.
hied. (Suppl.) 134:1228-1236.

Vlard, P. A., and J. W. Kibuka-Musoke.' 1969. Evidence for soluble
inmmne complexes in the pathogenesis of the glomerulonephritis

<>f quartan malaria. Lancet 1:283-285.

57
IL Weigle, W. O. 1961. Fate and biological action of antigen and
antibody complexes, Vol. 1., p. 283. ‘In_Taliaferro, W. H., and
J. H. Humphrey (eds.), Advances in immunology, Vol. 1. Academic

Press, N.Y.

58
Table 1. Average of percentage of parasitized erythrocytes (%PE), red
blood cell counts (RBC x 106), spleen volume in m1. , severity of kidney
damage (SKD), the titers of hemagglutinin for trypsinized erythrocytes
(HA), serum antigen (SA) and antibody to serum antigen (ABSA) in chickens

infected with 108 Plasmodium gallinaceum infected erythrocytes.

 

 

Days of Ave. % Ave. Ave. Spleen Ave. Ave. Ave. Ave.
Infection P.E. RBC x 106 Vol. m1. SKD HA SA ABSA
O O 2.45 1.8 39.3 0 O 0
l 2.75 2.16 3.9 130.7 0 8 O
2 15.75 1.92 5.6 209.0 16 64 16
3 44.50 1.93 7.2 202.7 32 128 128
4 69.75 1.85 4.6 261.0 128 32 256
5 60.00 1.01 5.3 274.0 16 8 256

Table 2 .

59

Averages of percentage of parasitized erythrocytes (%PE), red

blood cell counts (RBC x 106), spleen volume in ml., severity of kidney

damage (SKD), the titers of hemagglutinin for trypsinized erythrocytes

(HA),

infected with 10

6

Plasmodium gaZZinacewn infected erythrocytes.

serum antigen (SA) and antibody to serum antigen (ABSA) in chickens

 

 

Days of % Spleen

Infection 9.1:. RBC x 10 V01. m1. SKD HA SA ABSA
o 0 2.68 1.5 36 0 0 0
3 0 2.60 2.0 165 16 16 0
5 1.0 2.25 1.67 161 8 128 8
7 61.0 1.61 1.60 257 16 1024 256
9 61.3 1.29 2.67 300 8 64 128
11 76.3 0.92 6.70 302 64 128 [256
13 47.3 1.03 5.80 327 I6 128 1024
16 0 1.49 4.80 294 8 0 128

60

Table 3. Average red blood cell counts (RBC x 106) on normal chickens

injected with the plasma of chickens with acute Plasmodium gallinacewn

infection (Exptl.) and normal chickens injected with plasma of normal

birds (Control).

 

Days aft er

6

Average RBC x 10

% RBC

 

Injection Control Exptl. lost D.F. t. p.
0 2.71 i 0.14 .67 i 0.17 -1 6 0.30 N.S
l 2.67 i 0.11 .68 i 0.12 31 6 11.49 <0.001
2 2.66_+0.l7 .58 i 0.18 41 6 8.56 <0.001
3 2.69 i 0.15 .83 i 0.05 27 6 10.28 <0.001
4 2.78 i 0.10 .79 i 0.07 36 6 14.97 <0.001
5 2.73 i 0.17 .74 i 0.16 36 6 8.31 <0.001
6 2.63 i 0.12 .87 i; 0.20 28 6 6.31 <0.001
7 2.60 i 0.13 .91 i 0.25 26 6 4.76 <0.005
8 2 67 i 0.04 .02 i 0.09 24 6 12.00 <0.001
9 2 71 i 0.11 .28 i 0.25 16 6 3.03 <0.025
10 2.62 i 0.06 .30 i 0.25 12 6 2.42 N.S

Table 4 .

with "eluate" from P. gallinaceum infected erythrocytes and normal

61

chickens injected with "normal" red cell eluate.

Red blood cell counts (RBC x 106) of normal chickens injected

 

Average RBC x 10

6

 

Days after 7. RBC

Injection Control Exptl. lost D.F. t. p.
O .89 i 0.27 .94_+0.19 -1 8 0.35 N.S.
l .21 j; 0.50 .23 i 0.10 30 8 4.22 <0.005
2 .09 -_l-_ 0.29 .17 i 0.07 30 8 6.70 <0.001
3 23 j; 0 4O .34 i 0.13 30 8 4.74 <0.005
4 03 i 0.25 .67 i 0.23 13 8 2.38 <0.050
5 83 i 0 30 06 i 0.22 25 7 4.18 <0.005
7 82 i 0 35 15 j; 0 43 23 8 2.64 <0.050
9 97 j; 0 18 44 i O 62 9 8 1.81 N.S.

0.1-turn .Inn

 

 

62

 

 

 

 

 

aNa oma boa «om

oMm mwmum><

 

 

 

mma mama 5mm mama
«ma qqma 0mm mmmm
so mama mam mama
mqa mama mam nmmm
Nqa coma mNa coma ooa ooam emu mmmm
Nma mama oma snma an ommm ohm comm
oaa Numa ama qua moa . comm oam NmmN
aoa moma aoa Osma am mmmm mmm ammm
Ba . oz 05 . 62 new . 62 0m . oz
aoxoano coxoaeo omxoano aoxoaeo
mammam ameuoz mammam mooaumam: mammam anemoz mammam mooaumamz
coxoanu omuo>ooom amxuaeu woum>oomm coxoano amauoz

carcass amenoz

.maumama mason nuaB mcmxoano mo mammac mo coauomhaa mooco>mhuca umumm muse: «N SSmuumwNNum

ExmeosmUNm Sosa u0u0>000u mcoxoanu :a one mcoxoaeo amauoq ca AQMmV mmmmman momma: mo kuaum>mm .m magma

 

.__ —- ._-._.—-—————--~———.- -—

63

Figure 1. Nephritis in acute Plasmodium gallinaceum infections of
chickens.

A. Nephron (glomerulus and adjacent proximal convoluted tubules)
of normal chicken kidney. Note the size relationship of the glomerular
tuft and Bowman's space. Adjacent capillaries are patent and erythro-
cytes are clearly visible within the lumens. The lumen of the adjacent
convoluted tubules is patent and the tubular epithelium is intact. In
estimating severity of kidney damage (SKD) this nephron would be evalu-
ated as zero (H and E, 250x).

B. Nephron of a kidney taken from a malarious chicken at the peak
of the parasitemia-anemia crises. Note that capillaries adjacent to
the glomerulus are not obvious, the tuft completely occupies Bowman's
space, there is an increase in the number of mesangial cell nuclei, and
the lumen of most of the adjacent convoluted tubules is obliterated. A
nephron in this condition was given an SKD evaluation of 4 plus (H and
E stain, 400x).

C. Section from the proximal convoluted tubules of a kidney
taken from a malarious chicken at the peak of the parasitemia-
anemia crises. Note that the tubule (top right) contains a hyaline
cast. The tubule in the center contains a cast consisting of cellular
debris. The basement membrane of several of the tubules has separated
(blistered) from the tubular epithelium. Interstitial edema is also

evident (Giemsa stain, 250x).

 

64

 

Figure l

 

65

Figure 2. Nephritis in chickens induced by injection of plasma of
chickens with acute Plasmodium gallinaceum infection.

A. A nephron (glomerulus and adjacent proximal convoluted tubules
of a kidney taken from a chicken 24 hours after injection of malarious
plasma. Note that the glomerular tuft completely occupies Bowman's
space; however, hypercellularity is not evident. The tubule at the top
left appears necrotic. Epithelium of other tubules is swollen and the
lumen of the two lower tubules appears to contain casts. A nephron in
this condition was given a severity of kidney damage (SKD) evaluation of
4 plus (Mallory's stain, 250x).

B. Nephron of a kidney taken from a chicken 24 hours after injec-
tion of normal chicken plasma. While the nephron appears slightly edema-
tous, it does not differ remarkably from the nephron shown in Figure 1A.
The SKD was estimated at 1 plus (H and E stain, 250x).

C. Section from area of distal convoluted tubules of a kidney
taken from a chicken 24 hours after injection of malarious plasma. Note
that 3 tubules at the center are filled with hyaline casts. The epi-
thelium of other tubules is swollen (Giemsa stain, 250x).

D. Nephron of kidney from a chicken that had recovered from acute
P. gallinacaum infection, 24 hours after injection of malarious plasma.
The semilunar crescenting, the fibrinous adherence of the tuft to Bowman's
membrane, and the evidence of desquamation of tubular epithelium are
characteristic of the nephron of recovered chickens (H and E stain,

250x).

   

Figure 2

Article 2

PATHOGENESIS OF ACUTE AVIAN MALARIA
113. A STUDY OF ANTIGENS AND ANTIBODIES ASSOCIATED WITH ANEMIA

OF ACUTE PLASMODIUM GALLINACEUM INFECTION OF CHICKENS

Jiya L. Soni and Herbert W. Cox
Department of Microbiology and Public Health,
Michigan State University

East Lansing, Michigan 48823

(T0 be submitted to The American Journal of Tropical Medicine & Hygiene)

PATHOGENESIS OF ACUTE AVIAN MALARIA
II. A STUDY OF ANTIGENS AND ANTIBODIES ASSOCIATED WITH ANEMIA

OF ACUTE PLASMODIUM GALLINACEMM INFECTION OF CHICKENS

*
Jiya L. Soni and Herbert W. Cox

Department of Microbiology and Public Health,
Michigan State University,

East Lansing, Michigan 48823

U.S. State Department, Agency for International Development
Fellow. Present address: College of Veterinary Science and Animal
Husbandry, Jawaharlal Nehru Agricultural University, Jabalpur,

Madhaya-Pradesh, India.

67

68
ACKNOWLEDGEMENTS
Research here reported is from a thesis entitled, "Pathogenesis of

Acute Avian Malaria,"

submitted by the principal author, in partial
fulfillment of the requirements for the Doctor of Philosophy degree at
Michigan State University. Participation in this program was made
possible by a fellowship from the U.S. State Department Agency for
International Development.

This research was supported in part with funds from Grant No.
AI-08508 from the National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland, and with support
from the Michigan Agricultural Experiment Station. This publication is

Journal Article No. from the Michigan Agricultural Experiment

Station.

ABSTRACT

The anemia of acute Piasmodium gaZZinaceum malaria of chickens had
been found to be in part mediated by substances in the plasma of malarious
chickens. Injections of malarious plasma into normal chickens produced
a 30% reduction in red blood cell counts within 48 hours which persisted
for more than a week. Injections of hypertonic salt solution eluates
from cells of malarious blood produced a similar anemia. Serologic
study of plasma implicated two mechanisms, a cold-active IgM class of
hemagglutinin and a soluble globulin associated serum antigen and its
antibody. The serum antigen was shown to be serologically unrelated to
parasite antigen and similar, if not identical, to serum antigen elabor-
ated during red blood cell infections with parasites other than those
of the genus Plasmodium, e.g., Babesia. Study of cell eluates from
chickens made anemic by plasma injections revealed the presence of both

serum antigen and its antibody. Immunofluorescent study revealed that

 

 

69
the blood cells of these birds reacted strongly with conjugates of
antibody prepared in chickens to serum antigen from rats with acute
babesiosis, and from chickens with acute P. gallinaceum malaria. The
cells did not react with conjugates of antibody to P. gaZZinaceum para-
site antigen. Strong reactions with anti-chicken 7S globulin suggested
that antibody was also present on the surface of the blood cells.
Immunofluorescent activity was substantially reduced three days after
injection when anemia was maximal. These experiments lead to the con-
clusion that the anemia of acute P. gdZZinaceum malaria of chickens is
in part an immune complex disease, and that serum antigen, which was
serologically unrelated to parasite antigen, was a principal causal

ingredient.

INTRODUCT ION

Immune mechanisms have been implicated in the anemia of acute
haemosporidian infections. The association of cold active hemagglutinin
for trypsinized erythrocytes with anemia, splenomegaly, and phagocy-
tosis of erythrocytes by splenic macrophages in acute malaria and
babesiosis of rodents led to the suggestion that autoimmune-like
medhanisms might have been in part responsible (1,2). Anemia follow-
ing the injection of globulins from animals with acute malaria or
babesiosis further indicated that there were anemia inducing substances
in the blood of such animals (3,4). This observation was confirmed
by injecting normal chickens with plasma of chickens with acute Plas-
modium gaZZinaceum infection. A 30% reduction in the red cell counts
was achieved within 24 hours and the birds did not recover from this
anemia until after 8 days. It was further indicated that anemia inducing

substances in the plasma had combined with red cells, by producing a

70
similar anemia with material leached from blood cells of malarious
chickens (5) .

Cold-active hemagglutinin was associated with acute avian malaria
and was identified as cold-active IgM (6). These observations were
confirmed by Soni and Cox (5). It was also found that the hemagglutinin
recovered from the plasma of chickens with acute P. gallinaceum infection
by absorption with, and elution from, trypsinized erythrocytes, caused
anaphylactic-like shock when injected into normal chickens. Plasma
samples collected 24 hours later showed signs of hemolysis and the red
cell counts of the birds were reduced by 25%. When plasma that had been
absorbed free of hemagglutinin was injected into normal chickens, it
too caused a 25% drop in red cell counts. Thus it was indicated that
there was more than one anemia inducing factor in the plasma of chickens
with acute P. gallinaceum malaria (7).

In earlier work showing that serum from animals with acute malaria
and babesiosis contained anemia inducing substances, the only identified
component injected had been an immunogen termed serum antigen. With
fluorescein conjugated antibody to serum antigen, it was shown that the
antigen had combined with the red cells of the injected animals and
had caused them to be removed from the circulation (3,4). The means
whereby serum antigen could combine with erythrocytes was not indicated.
An insight was furnished by the observation that the anemia of acute
Babesia rodhaini infections of mice was better correlated with the con-
current presence of serum antigen, and antibody to serum antigen, than
it was to parasitemia or to the titers of hemagglutinin for trypsinized

erythrocytes (8).

71
Thus a mechanism suggested by Dixon (9) that involved the binding
of soluble antigen-antibody complexes to erythrocytes to act as opsonins
was indicated as a possible anemia inducing factor. The antigens and
antibodies in the blood of chickens with acute P. gallinaceum infection
have been identified and those that react in vivo with normal erythro—
cytes have been indicated. The details of this study are presented in

this communication.

MATERIALS AND METHODS

Animals and Infections: The source of the P. gaZZinaceum infec-

 

 

tion, the chickens used, methods for maintenance, experimental infections,
and the parameters for evaluating infection and pathogenesis have been
described elsewhere (5).

Chickens infected with P; gallinaceum were exsanguinated at the
peak of parasitemia and the blood was mixed 10:1 with heparinized saline
(100 units Sodium Heparin to 1.0 m1 of 0.78% NaCl solution) as described
(5). After centrifugation at 800 g for 10 minutes the supernatant
plasma was removed and stored at -18 C. The sedimented cells were
resuspended and washed twice with saline (0.78% NaCl solution) by
centrifugation at 800 g and resuspension in saline. The washed cells
were then leached with hypertonic NaCl (1.2%) solution (5). This leaChed

material was stored at -18 0.

Preparation of Parasite Antigen: Washed cells of malarious chicken
blood were treated to liberate parasites by methods modified from those
of Sherman and Hull (10) (see Flow Chart). After concentration and
tests for antigen activity, the soluble parasite antigen (PA) was

stored at -18 C.

72
The concentrated PA was further purified by column chromatographic
methods using Sephadex G-200 as described below. Fractions from the
column were pooled as shown (Figure 2). After concentration by poly-
ethylene glycol treatment, the pools were tested for antigen in double
diffusion in gel tests using plasma of recovered chickens. Active

fraction pools were stored at -18 C.

Preparation of Serum Antigens: Serum antigen was detected in
Plasma of malarious chickens by means of a tube bentonite flocculation
(TBF) test using bentonite suspension treated with globulin of rats
recovered from B. 17065162737273 as described by Thoongsuwan and Cox (8).
This test was later modified to use bentonite treated with globulin of
Chickens that had been immunized with serum antigen of rats with acute
3- rodha’im: infection (ABr).

Plasma with serum antigen was clarified by centrifugation at 2000
8 for 30 minutes at 5’ C. The plasma was then subjected to column chroma-
tc>graphy using Sephadex G-200 as described below. Fraction pools
ob tained as shown in Figure 2 were concentrated by ultrafiltration in
dialysis tubing under negative pressure, or by polyethylene glycol
dehydration. After tests for activity against ABr, the active frac-
t:‘~<>‘l:ls were stored at -18 C.

Attempts were made to further separate serum antigen fractions by
Ixtecipitation with sodium sulphate and dextran sulphate following

methods described by Lykins at al. (11).

Preparation of Leach Antigen: Antigens were eluted from the
washed cells of malarious chicken blood with hypertonic 1.2% NaCl solu-
tion as described (5). These preparations were then subjected to column

chli‘omatography using Sephadex G-100 as described below. Fraction pools

73
as shown in Figure 2 were tested for antigen activity in gel tests using
ABI‘ and plasma of chickens recovered from P. gallinaceum infection.

Fractions with antigen activity were stored at -18 C.

Column Chromatography Methods: Molecular sieving methods used

were Sephadex G-200 or G-100, prepared in borate buffered NaCl solu-
tion, pH 8.2, with an ionic strength of 0.16 (BBS) as described by

Benedict (12). The column size was 100 x 2.5 cm with a packed column

size of 90 x 2.5 cm. The flow rate was adjusted to 15 ml per hour and

the collector was set for 3.0 m1. All molecular sieving was done at

a constant room temperature of 22 C. Four ml volumes, containing 160

mg of protein, were equilibrated by dialysis overnight against BBS.

The sample was then mixed with 0.16 gm of sucrose and applied to the
CO lumn. Fractions were tested for absorbance of light at 280 nm and
8553’ propriate fractions were pooled (Figure 2). Each pool was dialyzed

in 1:100 BBS overnight, and then concentrated by 1yophilization, ultra-

filtration, or dehydration with polyethylene glycol.

Double Diffusion in Gel Test Methods: Modifications of the gel

diffusion test methods of Ouchterlony (13) as described by Lykins et
a: . (11) were employed, using as a base 1% Colab Ion Agar No. 2. The
agar gel was made in 1.5 M NaCl to test for serum antigen, and in
veronal Acetate buffer at pH 8.6, ionic strength of 0.1, for other

aantigens. Sodium azide to give a concentration of 0.02% was added to

1:l'le gel to prevent contaminant growth. A11 gel slides were incubated

at 22 C for as long as 5 days and multiple feedings of wells was done
as needed. Precipitin lines were stained by methods of Uriel (14).
Photographic records were made of reactions in gel by photographing

either the fresh wet preparations, or stained dried slides.

 

74

Immgoelectrgghoresis (IEP) Methods: IEP was accomplished with a

 

Gelman Instrument Co. apparatus and Gelman's high resolution Tris-
Barbitol-Sodium Barbitol buffer, pH 8.8, with an ionic strength of

O . 05. Slides were prepared from 1% Colab Ion Agar No. 2 in Gelman's
buffer. Test slides were exposed to 18 ma of current for 65 minutes.
After electrophoresis, the slides were reacted against anti-chicken
serum or anti-chicken globulin for 48 to 72 hours at 22 C. The slides

were washed and stained as described by Uriel (14).

Disc Electrophoresis (DEP) Methods: The physicochemical nature of

 

blood fractions showing antigen activity was determined by disc electro-
pho resis using modifications of methods described by Davis (15). The
running gel consisted of 7% cyanogum in Tris-HCl buffer pH 8.6 which was
Polymerized by addition of TEMED (N,N,N',N'-Tetramethylethy1ene) and
Amtnonium persulphate (AP). The spacer gel consisted of 4% cyanogum in
TT53LS—HC1 buffer, pH 8.2, polymerized with TEMED and AP. Samples to be
run were made up to a 4% sucrose solution and 20 111 were applied to the
SPaCer gel, along with a drop of Bromophenol blue to serve as tracking
dye, The current was applied at 5 ma per tube until the migration was
comPlete. Discs were stained differentially for protein, lipids, carbo-
hydt‘ates, and for deoxyribonucleic acid as described by Turner and

McGregor (16) .

Qibe Bentonite Flocculation (TBF) Test for Antigens and Anti-
M: Suspensions of sized bentonite particles have been used to
titTate antigens by treating them with antibody and for titrating
antibody by treating the bentonite with antigen (5,8) . The methods for
Preparing stock bentonite suspension, treating the bentonite with anti-

gene or antibodies, and accomplishing the TBF test have been described

75
(5 , 8,17). In the present work, the TBF test was used to titrate antigens
present in material leached from blood cells, antigens prepared from
parasites, and serum antigen. One tenth ml of globulin from immunized
chickens, diluted 1:20 with phosphate buffered 0.85% NaCl solution,

pH 7.2 (PBS), was used to sensitize 10 ml of bentonite suspension.

Preparation of Antiserum: ABr was prepared in chickens by immuniz-
ing them with serum antigen from rats with acute B. rodhaini infection.
Each chicken was given 0.5 ml of plasma mixed with 0.5 ml of complete
Freund's adjuvant injected at multiple sites, followed by similar
injections one week later. After one week, a test bleeding of the
chickens was made and they were given a 3rd injection of the antigen
with adjuvant. Two weeks later the birds were exsanguinated and the
Plasma recovered. Plasma from these birds was tested for antibody in
8&1 diffusion tests against serum antigen bearing plasma of chickens
With acute P. gaZZinaceum infections. After clarification, the plasma
was stored at -18 C.

Antisera to each of the purified antigens from the blood of malar-
ions chickens was prepared in chickens by immunizing them, each with
1‘0 mg of antigen in 0.5 m1 of saline mixed with an equal volume of
comPlete Freund's adjuvant as described above. These birds were exsan-
gu:Lrlc'ftted 2 weeks after the 3rd injection and the plasma tested against

the respective antigen in gel diffusion tests. They were also tested

for specificity against the homologous and heterologous antigens in gel

and TBF tests.

Antisera to Normal Chicken Serum and to Normal Chicken Globulin:

 

Bl°°d from normal chickens was withdrawn and allowed to stand overnight

at 5 C. The next day it was centrifuged at 2000 g for 30 minutes and

76

the supernatant serum was recovered and stored at -18 C. A portion of
the serum was precipitated with an equal volume of saturated ammonium
sulphate and the recovered globulin was dialyzed free of sulphate as
described (5). One mg of serum, or 1.0 mg of globulin, in 0.5 ml of
O - 787. NaCl solution was mixed with an equal volume of complete Freund's
adjuvant and each was given by multiple site intramuscular injection to
rabbits. The rabbits were given a second injection a week later and
after another week a trial bleeding was made. Sera from these rabbits
reacted strongly in gel tests with chicken serum, or with whole globulin.
The rabbits were given an additional injection and were exsanguinated
the following week.

Antiserum to chicken 78 globulin was obtained from Nutritional

Biochemical Corporation, Cleveland, Ohio.

Fluorescein Isothiogyanate (FITC) Conjugation of Antisera: Antisera
'50 be used for fluorescent antibody testing were conjugated with FITC.
GlObulins obtained from sera or clarified plasma, by 507. ammonium sulphate
Precipitation, were dialyzed against BBS for 2 days with a change of
buffer after 24 hours, to remove ammonium sulphate. The protein concen-
tration of the globulins was determined as described by Lowery et al.
(18) and was adjusted to a concentration of 20 mg per ml by addition of
'852 NaCl solution. FITC conjugation was achieved by methods described
by Cherry et al. (19). The conjugate was then tested in gel against

antigen to ensure activity before it was stored in small aliquots at

‘70 c.

fluorescent Antibody Tests and Microghotography: Blood slides from
malarious chickens, chickens injected with plasma of malarious chickens,

and control birds were prepared and stored at -70 C as recommended by

77

Sulzer and Wilson (20). They were gradually equilibrated to room
temperature by holding the slides at -20 C for 30 minutes, at 4 C for
30 minutes and then at room temperature before the slides were fixed in
Acetone for 10 minutes. The slides were rinsed in PBS, pH 7.0. Direct
staining was accomplished as described by Ward and Conran (21).

Fluorescence was determined with a Carl Zeiss Fluorescope equipped
with an Osram HBC 200 Mercury lamp using excitor filter II BG 13 and
barrier filter 50/44. Microphotographs were taken with TRI-X-Pan Kodak

film at an exposure time of 30 and 60 seconds.
EXPERIMENTS AND RESULTS

Tests of Components of Blood from Malarious Chickens for Antigens:
The plasma, the material leached from the washed blood cells with hyper-
tonic NaCl solution, and the material extracted from liberated parasites
each reacted with plasma of chickens that had recovered from acute P.
gallinaceum infection (Figure l-l). The plasma, here designated as serum
antigen (SA), the leach antigen (LA), reacted with globulin of chickens
immu‘II'Lszed with plasma of rats with acute B. rodha‘iml (ABr). However,
the parasite antigen (PA) did not react with ABr, (Figure 1-2).

In further tests the leached material reacted in gel tests with
Plasma of recovered chickens, ABr and antibody to serum antigen from
Ch1<=1<ens (ASA) but did not react strongly with antibody to parasite
antigen (APA (Figure 1-3). The reaction with serum antigen from rats
With acute B. rodhaini (BrA) indicated the presence of antibody to serrml
antigen, as did also the reaction with serum antigen from malarious

Chickens (SA2), and the reaction with anti-chicken 7S globulin (Figures

1‘4’ 1‘5: 1‘6) 0

 

78

In immunoelectrophoresis, tests of LA with anti—chicken serum, the
presence of 198 and 7S globulins, along with albumin, was indicated.
Irmnunoelectrophoresis of leach from normal blood cells, tested with
anti-chicken globulin, revealed only traces of protein (Figure 1-7).
However, disc electrophoresis of both leach preparations revealed the
presence of a variety of bands taking protein stain. One band in the
19S globulin range distinguished the leach from malarious blood cells
from that of the normal cells (Figure 1-8).

After column chromatography of each of the crude components from
malarious blood, fraction pools as indicated in Figure 2 were made and
tested in gel against plasma of recovered chickens. Pools 1, 2 and 3
of the fractionated SA, Pools 2 and 3 of leached material, and all pools
collected from the column for parasite extract reacted. Thus it appears
that antigen(s) present in plasma was restricted to the 198 and 7S
PrOtein peaks. Antigen activity in pool 2 of the leached material was
eluted with the globulin class proteins. However, the activity in pool
3 Was associated with proteins of lower molecular weight. The results
from the tests of column fraction pools of parasite antigen are unclear.
Antigen activity was found in all pools, including the 5th, which did
not Show absorbance at 280 nm.

An attempt was made to further purify the active SA fractions by
precipitating each with Dextran Sulphate. The supernatant was then

\
precipitated with 10% sodium sulphate. In each fraction pool, the '
mater’ial precipitated with the sodium sulphate (SA-l) and Dextran Sul-
Phate (SA-2) reacted with both recovered chicken globulin and with ABr.

SA—z, but not SA-l, reacted in gel tests with antibody to parasite

antigen .

79

Antibody prepared in chickens to the various column fraction pools
was used to test for monospecific antigen and antibody in both gel and
TBF tests. The tests indicated that the only pure antigen and antibody
obtained was SA-l and its antibody. While the leached pools contained
serum antigen, it was also evident that they contained parasite antigen.
Cross reactions with parasite antigen pools and their various antibodies
did not further clarify the status of parasite antigen, except that they

did not react with anti-SA-l.

Antibodies Found in Blood Durig the Course of Acute Avian Malaria:

Chickens 6-8 weeks old were inoculated intravenously with 108

P. gaZZina-
,eeum parasitized erythrocytes and control birds were given an equivalent
inj ection of normal chicken cells. Birds of both groups were examined
daily for RBC counts, the percent of erythrocytes parasitized (ZPE) was
determined, and blood plasma samples were taken for serologic study.
These were tested for antibody to SA, LA, and PA using the TBF test with
ben tonite sensitized with SA-l, LA-2, and PA—é. The results of these
Studies are shown in Figure 4. Parasitemia was first patent on day 2 and
reaChed its peak on day 8. The RBC counts did not decrease sharply until
3“- ter the 5th day of infection and between the 5th and 8th days fell
from approximately 2.25 x 106 to 0.8 x 106. None of the plasma samples
collected on days 1 and 2 reacted with antigen sensitized bentonite.
H°Wever, those collected thereafter reacted with all three antigen prepa-
lrations, reaching their highest titers between the 5th and 8th days.

In general, the appearance of antibodies and high parasitemia were corre-

lated with anemia .

80

lmmuno_logic Study of Anemia Inducing Factors in Plasma from Chickens
Bil-th Acute P. gaZZinaceum Infections: For these experiments, 4 chickens
were given 4 ml of plasma from chickens with acute P. gal Zinaceum infec-
tion by intravenous injection. Four other birds were inoculated in the
same manner with 4 ml of plasma from normal chickens. In both cases- the
protein content of the inocula was estimated to be approximately 164 mg.
Red blood cell counts were made prior to injection, and subsequently at
3, 6, 12, 24, 48, 72, 96 and 120 hours after inoculation. The results
of these counts are presented in Table l. The cell counts were reduced
by 21% after 3 hours and to 347. at 24. Recovery from the effects of the
injections was not evident until after 72 hours. Statistical analysis
indicated that the differences in the cell counts were highly
Significant.

Another group of 4 experimental and 4 control chickens were given
Plasma injections as described above. One experimental and one control
bird were sacrificed by exsanguination at 6, 12, 18 and 24 hours after
inj ection. After centrifugation, the plasma was recovered and the cells
were divided into equal portions. To one portion, 0.787. NaCl solution
was added to suspend the cells to original volume. After gentle agita-
tion of the suspension, the supernatant was removed and held for study.
TheSe cells and the portion of unwashed cells were subjected to leaching.
The plasma, the cell washings, and the material leached from both cell
I3°"-‘t:l.ons were then tested for antibody using the TBF test with bentonite
sensitized with serum antigen from rats with acute B. rodhaini, and with
globIllin of chickens with acute P. gallinacewn, infection. The prepara-
tions were also tested for serum antigen with bentonite treated with
AB": and with globulin of recovered chickens. Results of the tests are

preseflted in Table 2. Titers of the antigen and antibodies did not

 

 

81
differ, regardless of the origin of serum antigen, or antibody to serum
antigen, used to sensitize the bentonite. Serum antigen and its antibody
were present in the plasma of the recipient chickens and persisted
throughout the 24 hours of the experiment. The antigen and antibody
were also present in the wash solution, and in the material leached from
both washed and unwashed cells. It was also noted that antigen titers
tended to be higher than those of antibody throughout the experiment.
Tests of materials from chickens injected with normal plasma were
negative.

Blood smears were prepared from chickens after injection of plasma
from.malarious birds and were incubated with FITC conjugated with ABr,
anti-SA-l, anti-PA, and anti-chicken 7S globulin. In slides prepared
up to 24 hours after injection, fluorescence was observed about the
periphery of numerous cells. Immunofluorescence was observed with ABr,
anti-SArl, and anti-7S, but not with anti—PA conjugate. Blood cells

from chickens injected with normal plasma did not react (Figure 5).

DISCUSSION

This study confirmed the observation that plasma of chickens with
acute P. gallinaceum infection contained both antigen and antibody, and
that injection of such plasma into normal chickens caused anemia. It
also confirmed the observation that material leached from washed blood
cells of malarious birds produced anemia in a similar manner (5).

Serologic study of material leached from the washed cells of
malarious chickens, and from washed blood cells of chickens made anemic
with plasma injections, indicated that both antigen and antibody were
present. That antigen and antibody had combined with the cells in viva

was indicated by fluorescent antibody staining of blood cells withdrawn

 

82
from the recipient birds, with conjugated antibody to the antigen and'
‘with conjugated antibody to chicken 73 globulin. Since the number of
cells showing fluorescence was great soon after injection of malarious
plasma and was considerably diminished after 3 days, it is suggested that
reacting cells had been removed from the circulation.

From the data it is suggested that malarial anemia may in part be
mediated by complexes of soluble antigens and antibodies as was suggested
by Dixon (9).

To identify the antigens and antibodies responsible for anemia, an
attempt was made to detect all of the antigenic substances that were
present in the blood of malarious chickens. In addition to the plasma,
and the material leached from the washed cells, antigens were extracted
from parasites (PA) that had been liberated from infected erythrocytes.

Column chromatographic and precipitation treatment of plasma from
malarious chickens revealed the presence of two fractions with antigenic
activity which were termed serum.antigen, SA-l and SA—2. Both migrated
through Sephadex G-200 along with the 198 and 7S globulin fractions.

The SA—l fraction reacted in serologic tests only with plasma of
chickens recovered from malaria, anti-SA—l, and with the plasma of
chickens immunized with plasma of rats with acute B. rodkaini infection
(ABr). SA-2 also reacted with these antibodies, but also reacted with
antisera to PA.

Antigen in the material leached from cells of malarious birds was
partly associated with globulin fractions, but was also associated with
lower molecular weight proteins present in the leach. Two column frac-
tion pools with antigen activity were obtained, LA-2 and LA-3, both of
which reacted with plasma from recovered birds and ABr. LA-3 also
reacted with antibody to PA, indicating that a mixture of antigens was

present.

83
Attempts to obtain pure antigens from extracted parasite material

were not successful. All fractions collected after column chromatography

reacted with plasma of recovered chickens; however, none of them reacted
with ABr or with anti-SA—l. Gel tests using various PA fractions, or

their antibodies, did not cross react with SA-l or anti-SA-l. It was

therefore indicated that there were two distinct classes of antigens

present in blood of the chickens: the PA class that was associated with

parasites, and the SA class which was without parasite species

specificity.
Plasma samples taken daily during the course of P. gaZZinaceum

infection were tested for antibody using SA—l, LA-2, and PA. Antibody

to each of the antigens was detected on the 4th day and persisted through-

out the course of the experiment. However, except for the evidence for

the presence of PA in the SA-2 preparation, the only antigen present in
the plasma from malarious blood was the globulin associated serum
antigen.

While antibody to PA could have reacted with infected cells to

cause them to be sequestered, it is unlikely that it had a role in
aIlemia from the injection of plasma of malarious birds, since antigen
for complex formation was not found in great concentration. It is
therefore suggested that in these eXperiments, globulin associated
SQrum antigen and its antibody were the principal reactants detected
111 plasma, and in eluates from blood cells of plasma injected chickens,
that could have caused immune complex anemia.

In other work attempting to identify the mediators of anemia in
el-tperimental malarial infections, Musoke (22) found that serum antigen

and its antibody could not have been implicated in anemia from Plas-

modium chabaudi infections of rats. Plasma of rats made anemic by

84
P. chabaudi infection contained a soluble antigen that was species-
specific for P. chabaudi and antibody to this antigen was also detected.
Both antigen and antibody were eluted from the washed blood cells of
anemic rats. It was suggested that immune complexes of this antigen
and antibody might have been instrumental in anemia. It therefore
appears that immune complexes, involving antigens other than serum

antigen, may have a role in malarial anemia.

Another immune mechanism associated with anemia of acute malaria
has been investigated. A cold-active hemagglutinin for trypsinized
erythrocytes was shown to be associated with the anemia of both acute
malaria and babesiosis of rats (1,2,22).

Barrett at al. (6) detected cold-active agglutinin in the blood
of chickens with acute P. Zophurae infection using trypsinized human
type "0" erythrocytes. It was felt that this work should be further
investigated since McGhee and Loftis (23) and Ludford et a1. (24) had
indicated that P. Zophurae at their respective laboratories was con-
taminated with an anemia inducing agent, duck infectious anemia (DIA)
Virus. Ludford et al. (25) found that P. Zophurae strains maintained
at four laboratories were contaminated, and suggested that DIA virus
In:Lght have been an unrecognized companion of P. Zophurae for many years.
The strain of P. gallinaceum used at this laboratory was tested exten-
S ively for DIA virus contamination and was found to be clean.

Cold—active hemagglutinin was detected in the blood of chickens
V"’Zi’Jh P. gaZZinacewn anemia with trypsinized human type "0" erythrocytes
as reported by Barrett et al. (6) and was generally associated with the
anemia of infection (5).

Injection of hemagglutinin absorbed from malarious plasma into

tlormal chickens produced both an anaphylactic—like reaction and anemia.

85
Plasma from which hemagglutinin had been absorbed also produced anemia.
Since the hemagglutinin did not react with other antigens, or antibodies
from blood of malarious chickens, it was considered to be an auto
sultibody-like IgM (7).

The presence, or absence, of a serum antigen that was common to
aucute babesial and malarial infections, and the role of such antigens
111 immunity, has become a controversy (26). Serum antigen was found in
ttie blood of monkeys with acute Plasmodium knowlesi malaria by Eaton
(27). The presence of such an antigen was confirmed by Cox (3), who
anlso reported that the antigen from.malarious monkeys conferred immunity
2111 rats challenged with P. berghei.

Serum antigen from the blood of horses, dogs, and rats with acute
1>zabesiosis each reacted in serologic tests with sera from recovered
Inrorses, dogs, and rats. The antigens from rats and dogs each conferred
resistance to challenge with both heterologous and homologous Babesia
Sipecies (4,28,29). Cox et al. (30) demonstrated with serological
“methods that the serum antigens associated with acute malaria and babesi-
Clsis were similar if not identical. This observation was confirmed
14ndependently by Ludford at al. (31). Cross protective immunization
‘Vrith serum antigen was demonstrated with serum antigen from rats with
bnabesiosis against malaria in ducks (32). Recovery from acute malaria
Qonferred resistance to babesiosis, and recovery from babesiosis con-
3f5erred resistance to malaria (33). This cross immunization between
lfileterologous species and genera of haemosporidian parasites has been
ltepeatedly confirmed (34).

We detected serum antigen in the blood of rats with acute B.
leodhaini infection and used this antigen to immunize chickens. Anti-

loody from these chickens reacted in serologic tests with the plasma of

86
chickens with acute P. gallinacewn infection. The purified SA—l frac-
tion from plasma of these chickens stimulated antibody in chickens that
reacted in serologic tests with the plasma of rats with acute B.
rodhaini. These findings should satisfy objections raised by Smith et
al. (26) concerning the existence or serologic cross-reactivity of
globulin associated serum antigens.

The origin of serum antigen and the means whereby it functions in
acquired resistance have not been satisfactorily demonstrated. Sibinovic
et al. (28) found the immunogenic component to be associated with
macroglobulin and that its sedimentation coefficient was approximately
223. Amino acid analysis and enzyme studies did not differentiate it
from host globulin. The results of the present experiments suggest
that the serum antigen from chickens with acute P. gaZZinaceum infection
is a globulin associated antigen and may be similar to the one described
by Sibinovic at al. (28) .

It is difficult to visualize how an antigen that is unrelated to
a parasite could stimulate acquired resistance to infection. However,
it seems clear that this antigen and its antibody reacted with erythro-
c)Vtes and caused a dramatic and rapid reduction in circulating red
bILood cells.

It is suggested that complexes of the antigen and antibody had
El(:ted as opsonin to cause the cells to be sequestered in the spleen,
0:- in other cases caused complement fixation and lysis of the cells.

8 ince this mechanism would involve infected cells, it could be speculated
1:I‘nat in a sermn antigen immunized animal, parasitized as well as normal
1‘ed cells might be removed from the circulation mor rapidly than they
would have been in nonimmune controls. Such a mechanism might contribute

in this nonspecific‘manner to recovery from acute malaria.

l.

87

REFERENCES
Cox, H. W., Schroeder, W. F., and Ristic, M. 1966. Hemagglutina-
tion and erythrOphagocytosis associated with the anemia of Plasmodium
berghei infections of rats. J. Protozool. 13:327-332.
Schroeder, W. F., Cox, H. W., and Ristic, M. 1966. Anemia, para-
sitemia, erythrophagocytosis and haemagglutinin in Babesia rodhaini
infection. Ann. Trop. Med. Parasitol. 60:31-38.
Cox, H. W. 1966. A factor associated with anemia and immunity in
Plasmodium knowlesi infections. Milit. Med. 131(Sup):ll95-1200.
Sibinovic, K. H., Milar, R., Ristic, M., and Cox, H. W. 1969. in
vivo and in vitro effect of serum antigens of babesial infection and
their antibodies on parasitized and normal erythrocytes. Ann. Trop.
Med. Parasitol. 63:327-336.
Soni, J. L., and Cox, H. W. l973a. Pathogenesis of acute avian
malaria. I. Immunologic reactions associated with anemia and
nephritis. In preparation.
Barrett, J. T., Rigney, M. M., and Breitenbach, R. P. 1970. Char-
acteristics of the hemagglutinin produced during Plasmodium
Zophurae malaria in chickens. Infection and Immunity 2:304-308.
Soni, J. L., and Cox, H. W. l973d. Pathogenesis of acute avian
malaria. IV. Anemia mediated by the cold-active agglutinin for
trypsinized erythrocytes from the blood of chickens with acute
Plasmodium gaZZinaceum infection. In preparation.
Thoongsuwan, S., and Cox, H. W. 1973. Antigenic variants of the
haemosporidian parasite Babesia rodhaini selected by in vitro
treatment with immune globulin. Ann. Trop. Med. Parasitol.

(In press).

10.

11..

12.

Jm3.

1u4.

88
Dixon, F. J. 1966. Comments on immunopathology. Milit Med. 131
(Sup):1233-1234.
Sherman, I. W., and Hull, R. W. 1960. The pigment (hemozoin) and
proteins of the avian malaria parasite Plasmodium Zophurae. J.
Protozool. 7:409-416.
Lykins, J. D., Smith, A. R., Voss, E. W., and Ristic, M. 1971.
Physical separation of three soluble malarial antigens from the
serum of chickens infected with Plasmodfium gallinaceum. Am. J.
Trop. Med. Hyg. 20:394-401.
Benedict, A. A. 1967. Production and purification of chicken
immunoglobulins, p. 229-237. .lE Williams and Chase (ed.) Methods
in Immunology and Immunochemistry. Vol. I. Academic Press, New
York.
Ouchterlony, O. 1953. Antigen and antibody reactions in gels.
IV. Types of reactions in coordinated systems. Acta Path. Micro-
biol. Scand. 32:231.
Uriel, J. 1971. Characterization of precipitates in gels: Color
reactions for the identification of antigen and antibody precipi-
tates in gels, Section l4El. .ln Williams and Chase (ed.) Methods
in Immunology and Immunochemistry. Vol. III. Academic Press, New
York.
Davis, B. J. 1964. Disc electrophoresis. II. Method and appli-
cation to human serum proteins. Ann. N.Y. Acad. Sci. 121:404—429.
Turner, M. W., and McGregor, I. A. 1969a. Studies on the immunol-
ogy of human malaria. I. Preliminary characterization of antigens

in Plasmodflum falciparum infections. Clin. Exp. Immunol. 5:1-16.

17.

113.

119.

21).

221”

212.

IZZL

89
Sibinovic, K. H., Ristic, M., Sibinovic, S., and Philips, T. N.
1965. Equine babesiosis: Isolation and serologic characterization
of a blood serum antigen from the acutely infected horses. Am. J.
Vet. Res. 110:147-153.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.
1951. Protein measurement with the Folin Phenol reagent. J. Biol.
Chem. 193:265-275.
Cherry, W. B., Goldman, M., and Carski, T. R. 1960. Fluorescent
antibody techniques in the diagnosis of communicable disease. U.S.
Public Health Service Publ. 729.
Sulzer, A. J., and Wilson, M. 1967. The use of thick smear antigen
slides in the malaria indirect fluorescent antibody test. J.
Parasitol. 53:1110-1111.
Ward, P. A., and Conran, P. B. 1966. Immunopathologic studies of
simian malaria. Milit. Med. 131(Sup):1225-1232.
Musoke, A. J. 1973. Immunologic and pathologic studies of infec-
tions with rat adapted Plasmodium chabaudi. M.S. Thesis, Michigan
State University.
McGhee, R. B., and Loftis, E. 1968. A filterable proliferating
factor simulating autoimmunity in malarious and non malarious
duckling. Exp. Parasitol. 22:299-308.
Ludford, C. G., Corwin, R. M., Cox, H. W., and Sheldon, T. A.
1969. Resistance of ducks to a Plasmodium sp. induced by a filter-
able agent. Milit. Med. 134(Sup):1276-1283.
Ludford, C. G., Purchase, H. G., and Cox, H. W. 1972. Duck
infectious anemia virus associated with Plasmodium Zophurae. Exp.

Parasitol. 31:29-38.

26.

27.

28.

29.

30.

31.

32.

33.

34.

90
Smith, A. R., Karr, L. J., Lykins, J. D., and Ristic, M. 1972.
Serum soluble antigen of malaria: A review. Exp. Parasitol. 31:
120-125.
Eaton, M. D. 1939. The soluble malarial antigen in the serum of
monkeys infected with Plasmodium knowlesi. J. Exp. Med. 69:517-553.
Sibinovic, K. H., MacLeod, R., Ristic, M., Sibinovic, S., and Cox,
H. W. 1967a. A study of some of the physical, chemical and sero—
logic properties of antigens from sera of horses, dogs, and rats
saith acute babesiosis. J. Parasitol. 53:919-923.
Sibinovic, K. H., Sibinovic, S., Ristic, M., and Cox, H. W. 1967b.
Immunogenic prOperties of babesial serum antigens. J. Parasitol.
53: 1121-1129 .
(30x, H. W., Milar, R., and Patterson, S. 1968. Serologic cross-
reactions of serum antigens associated with acute Plasmodium and
Babesia infections. Am. J- Trop Med. Hyg. 17:13-18.
Ludford, C. C., Hall, W. T. K., Sulzer, A. J., and Wilson, M.
1972. Babesia argentina, Plasmodium vivax, P. falciparwn: Anti-
genic cross reactions. Exp. Parasitol. 32:317-326.
Corwin, R. M., and Cox, H. W. 1969. The immunogenic activities
Of the non-specific serum antigens of acute haemosporidian infec-
tions. Milit. Med. 134(Sup):1258-1265.
Cox, H. W., and Milar, R. 1968. Cross-protection immunization by
Plasmodium and Babesia infections of rats and mice. Am. J. Trop.
Med. Hyg. 17:173-179.
Cox, F. E. G. l972a. Protective heterologous immunity between
Plasmodium atheruri and other Plasmodium spp and Babesia spp in

mice. Parasitology 65: 379-387 .

91

TABLE 1

Red blood cell counts (RBC x 106) of normal chickens after injection of

plasma from chickens with acute Plasmodium gallinaceum infection (Exptl.)

arid' plasma of normal chickens (Control) and the percent of RBC lost

(Z RBC lost).

 

 

Par t iculars Exp t1 . Control
Nurnber of chickens 4 4
Ave. 6 Ave. Z RBC t
Time after Injections RBC x 10 RBC x 10 lost value P
0 hours .08 i 0.27 2.97 i 0.59 -3.7 0.26 N.S.
3 hours .32 i 0.09 2.93 i 0.42 20.8 2.81 <0.050
6 hours .95 i 0.24 2.81 -_i-_ 0.30 30.6 4.43 <0.005
12 hours 88 i 0.15 2.81 i 0.26 33.1 6.06 <0.001
24 hours 84 i 0.17 2.80 i 0.30 34.3 5.43 <0.005
48 hours 88 i 0.54 2.86 i 0.17 34.2 3.42 <0.025
72 hours 00 i 0.16 2.83 i 0.09 29.33 8.57 <0.001
96 hours 91 i 0.23 2.84 i 0.10 32.7 7.19 <0.001
120 hours 11 i 0.50 2.73 i 0.13 22.7 2.61 <0.050

92

TABLE 2
Titers of serum antigen (SA) and antibody to serum antigen (ABSA) in
plasma (I), saline washings of blood cells (II), hypertonic saline leach—
of tlnwashed cells (III), and leach from washed cells (IV) from the blood
of chickens sacrificed at 6, 12, 18 and 24 hours after the injection of
4 ml' of plasma from chickens with acute Plasmodiwn gallinacewn malaria,
and in chickens injected with plasma of normal chickens.* Tested with
the tube bentonite flocculation test using bentonite treated with SA
from rats with acute babesiosis (R) and from chickens with acute malaria
(C) to detected ABSA, and ABSA of‘both rat and chicken origin to
detect SA.

Titers of SA and ABSA

6 Hrs. 12 Hrs. 18 Hrs. 24 Hrs.

SA ABSA SA ABSA SA AB SA SA AB SA

 

 

I c 64 32 32 o 64 32 64 32
R 64 64 32 16 64 32 64 64
II c 64 64 64 32 32 32 32 16
R 16 8 32 16 32 16 16 8
I II c 32 32 64 16 32 16 32 16
R 16 16 16 16 32 16 32 16
IV c 32 32 32 16 16 8 16 8
R 32 16 64 16 32 16 16 8
\ 4

 

a:
All tests made on blood from chickens injected with normal plasma were

negative .

93

Figure l. The serologic relationships of antigens found in the

blood of chickens with acute Plasmodium gaZZinaceum infection, and the

 

physicochemical properties of material leached from cells of malarious
blood.

1—1. The reactions in double diffusion in gel tests of malarious
plasma (SA), material leached from cells of malarious blood (LA), and the
material extracted from P. gallinaceum parasites (PA) with the plasma of

chickens recovered from P. gallinaceum infection (R).

1-2. The reactions of LA, SA, and PA with plasma of chickens that
had been immunized with plasma of rats with acute Babesia rodhaini infec-
tion (ABr). Note the line of identity between LA and SA and the failure
of PA to react with this antibody.

 

1-3. The reactions of LA with ABr, R, antibody to serum antigen
from chickens with acute P. gallinaceum infection (ASA), and antibody to
P. gaZZinaceum parasite antigen (APA). Note the failure of APA to react
with LA.

1-4. The reaction of LA with the plasma of rats with acute B.
rodhaini infection (BrA) and the failure of normal rat serum (NRS) to

react.

1-5. The reaction of LA with purified serum antigen from chickens
with acute P. gallinaceum infection (SA2) and failure of equivalent

plasma fraction of normal (N) chickens to react.

1r6. Tests of LA and material leached from cells of normal chicken
blood (N) with antibody to chicken 73 globulin.

1-7. Immunoelectrophoretic reaction of LA and normal chicken serum
with antibody to whole chicken serum (Top), and material leached from
cells of normal chickens with antibody to chicken globulin (Lower).

1-8. Disc electrophoresis of LA and material leached from cells of
normal chicken blood (N) stained for protein. Arrow indicates the pres-
ence of a high molecular weight protein in leach from infected cells

(I) which was not seen in the leach from normal cells (N).

94

 

Figure 1

95

Figure 2. Elution profile after column chromatography in Sephadex,
of plasma of malarious chickens, material leached from cells of malar-
ious chicken blood with hypertonic (1.2%) NaCl solution, and extract
from Plasmodium gallinaceum parasites. The columns were 2.5 x 100 cm,
the packed column size 2.5 x 90 cm, and columns were charged with 160 mg
protein. Elution was with borate buffered saline, pH 8.2, I = 0.16, the
flow rate was 15 ml per hour, and the fraction volume 3 m1/tube. All
columns were run at 22 C.

Pools of column fractions were made as shown. In tests for anti-
genic activity with plasma of chickens recovered from P. gaZZinaceum,
pools l, 2 and 3 of plasma protein, pools 2 and 3 of the leached

material and all pools of the parasite extract reacted.

OPWTCAL DEMENTY’AJ ZOOnuu

96

mcnouPOOLs
I I |2|3|4|5| 3|

'- PLASMA.
SEPHADEX 6-200

 

 

 

 

 

 

 

 

" LEACHED MATERIAL
SEPHADEX G-IOO

12
on
o.

c

| FRACTION Poms

lllzl 3 I 4 I 5 I
o .PARASITE EXTRACT
ssnmoex 0-200

0.

    

 

 

 

0'20466086100120140160
comm FRACTIONS (was NO.)

Figure 2

97

Figure 3. The relationships of parasitemia (average Z parasitized
erythrocytes), anemia (average RBC counts x 106) and average antibody
titers against serum antigen, leached antigen, and parasite antigen in
the blood of chickens throughout the course of Plasmodium gallinaceum
infection. Titers were determined with the tube bentonite flocculation
test. Serum antigen from rats with acute Babesia rodhaini infection,
as well as serum antigen from malarious chicken plasma,was used to test

for antibody to serum antigen.

40!

20

98

AVE. % PE

 

3

 

of AVE. RBC COUNTS I: I06
EXP.— CONT. ---

 

 

5000i

mm

500

I

IOO

I

50

 

AVE. ANTIBODY TITERS AGAINST
SERUM ANTIGEN

LEACH ANTIGEN - - --
PARASITE ANTIGEN"""°'

 

 

 

 

I 2 3' 4 5 5 7 8 9
ms AFTER INFECTION

Figure 3

99

Figure 4. Reactions of the blood cells of normal chickens that had
been injected intravenously with the plasma of chickens with acute Plas-
modium gaZZinaceum infections, with fluorescein isothiocyanate conju-
gates of antibody to serum antigen of rats with acute Babesia rodhaini
infection (ABr) prepared in chickens, antibody to serum antigen from
malarious chickens prepared in chickens (ASA), antibody to chicken 78
globulin (IgG) (commercial, prepared in rabbit), and antibody to P. gal-
Zinaceum parasite antigen (APA) prepared in chickens.

A. Reaction between blood cells and ABr 24 hours after injection
of plasma from malarious chickens. Cells from chickens injected with
normal plasma did not react.

B. Reactions seen with ABr conjugate 3 days after injection of
malarious plasma. Note that cells showing immunofluorescent activity
were reduced in number and appeared to be clumped.

C. Reaction of blood cells with ASA 24 hours after the chickens
had been injected with malarious plasma.

D. Reaction of blood cells with anti-7S globulin 24 hours after

the chickens had been injected with malarious plasma.

The APA conjugate did not react with cells from chickens injected

with malarious plasma.

Figure 4

 

101

Procedural steps for separating Plasmodium gallinaceum parasites from

erythrocytes and preparing parasite antigen (PA). Modified from methods

of Sherman and Hull (10).

1.

Draw blood from heavily infected chickens and add 1 m1 of heparin-
ized saline (100 I. U. sodium heparin/ml of 0.78% NaCl solution) per
10 ml of blood. Mix. Centrifuge at 800 g for 5 minutes. Discard
plasma.

Wash cells 3 times in modified Trager's buffer (MTB) QS to original
blood volume, with centrifugation at 800 g for 5 minutes. Remove
buffy coat after each washing.

Add 5 volumes of 1% saponin per volume of packed cells, mix and
incubate at 39 C for 15 minutes.

Centrifuge at 2000 g for 15 minutes at 4 C and discard the super-
natant. Wash the sediment 3 times with 5 volumes of MTB and centri-
fugation at 2000 g for 15 minutes at 4 C.

Mix sediment with 5 volumes of 1.0 M NaCl solution and incubate at
39 C for 30 minutes. Centrifuge at 2000 g for 15 minutes at 4 C.
Discard supernatant.

Wash sediment 2 times with 5 volumes of MTB and centrifugation as
in 5.

Mix sediment in 10 m1 of MTB and freeze-thaw 5 times by alternate
acetone-dry ice freezing and thawing at 37 C.

Centrifuge at 3000 g for 30 minutes at 4 C. Discard sediment.
Concentrate supernatant to 25% of original volume by dehydration

in dialysis tubing with polyethylene glycol flakes at 4 C.

Test for antigenic activity with globulin of chickens recovered

from P. gallinaceum infection in double diffusion in gel tests.

Article 3

PATHOGENESIS OF ACUTE AVIAN MALARIA
III. IMMUNOLOGIC MEDIATORS OF NEPHRITIS IN ACUTE PZASMODIUM

GALLINACEUM INFECTIONS 0F CHICKENS

Jiya L. Soni and Herbert W. Cox
Department of Microbiology and Public Health,
Michigan State University

East Lansing, Michigan 48823

(To be submitted to The American Journal of Tropical Medicine & Hygiene)

PATHOGENESIS OF ACUTE AVIAN MALARIA
III. IMMUNOLOGIC MEDIATORS 0F NEPHRITIS IN ACUTE PLASMODIUM

GALLINACEUM INFECTIONS 0F CHICKENS

*
Jiya L. Soni and Herbert W. Cox

Department of Microbiology and Public Health,
Michigan State University,

East Lansing, Michigan 48823

*

U.S. State Department, Agency for International Development,
Fellow. Present address: College of Veterinary Science and Animal
Husbandry, Jawaharlal Nehru Agricultural University, Jabalpur,

Madhaya 'Pradesh, India.

102

103
ACKNOWLEDGEMENTS
Research here reported is from a thesis entitled, "Pathogenesis of

Acute Avian Malaria,"

submitted by the principal author, in partial
fulfillment of the requirements for the Doctor of Philosophy degree at
Michigan State University. Participation in this program was made
possible by a Fellowship from the U.S. State Department Agency for
International Development.

This research was supported in part with funds from Grant No.
AI-08508 from the National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland, and with support
from the Michigan Agricultural Experiment Station. This publication

is Journal Article No. from the Michigan Agricultural Experiment

Station.

ABSTRACT

Nephritis associated with acute malaria has been suspected of being
an immunologic disease; however, the responsible antigens have not been
implicated. To get this basic information a study was made of the
kidneys and kidney functions of chickens with acute Plasmodium gaZZina-
ceum malaria. A study of the urinary wastes of malarious chickens
revealed that serum proteins were extravasated into the kidney to pass
in the urine. The leakage did not begin until globulin associated serum
antigen and its antibody were both detected in blood. This antigen and
its antibody were detected in the urine wastes for as long as antigen
was present in the blood. Afterwards, the presence of 7S globulin indi-
cated that protein leakage continued. A study of the materials extracted
from kidney tissues indicated the presence of serum proteins that were

not detected in tissues from normal chickens. Serum antigen and its

104
antibody were also present in these proteins. Frozen kidney sections
from malarious chickens were reacted with fluorescein isothiocyanate
conjugated antibody prepared in chickens against serum antigen from
rats with acute Babesia rodhaini infection (ABr), purified serum antigen
from malarious chickens (ASA), purified P. gallinaceum parasite antigen
(APA), and antibody to chicken 78 globulin prepared in rabbits (A7S).
With the ABr, ASA, and A78 a diffuse, granular "lumpy-bumpy" type of
immunofluorescence that has been described as characteristic for
immune complex nephritis was observed. Immunofluorescence with APA
conjugate differed in that the activity was localized to the glomerular
tuft. In the course of infection immunofluorescent activity was not
observed until both serum antigen and its antibody were detected in.
the blood, and activity with ABR, ASA and APA ceased on the day serum
antigen and parasitemia were no longer detected in the blood. Fluores-
cence with A7S continued undiminished well into the time the birds
appeared to have recovered from malaria. When sections from kidneys
of birds with nephritis induced by injections of malarious plasma were
studied, immunofluorescent activity of the diffuse granular "lumpy-bumpy"
type was observed with ABr, ASA and A78. No reaction was observed in
tests with APA. It was therefore concluded that parasite antigen played
no role in the nephritis induced by malarious plasma. Globulin associ—
ated serum antigen, which was serologically unrelated to P. gallinaceum
Parasite antigen, and which is associated with infections with parasites
Other than those of the genus Plasmodium, has been strongly implicated

as a causal agent in the acute nephritis associated.with.Ph gallinaceum

malaria of chickens.

105
INTRODUCTION

Glomerulonephritis associated with malaria is best known as the
chronic quartan malarial nephritis syndrome associated with Plasmodium
malariae infection in man. This disease is chronic and progressive in
nature, and eventually leads to renal failure with disease signs consistent
with those described by Bright.1 A nephritis with sudden onset, which
often resulted in renal shutdown, was less well recognized, and has in
recent years been found to be associated.with acute severe Plasmodium
falciparum infections, especially those that originated in South East
Asia.

In earlier studies it was suggested that factors such as anoxia
from acute anemia, or vascular failure due to the occlusion of the blood
vessels of the kidney,were the mediators of the disease.2’3’4

However, recent investigators have suggested that immunologic
mechanisms might have been involved. This suggestion has been based on
the observation that immune substances in the form of IgG, IgM, and
B C globulin have been seen as deposits in kidney sections stained with

l
fluorescein iSOthiocyanate (FITC) conjugated anti-globulin.5’6’7

Ultra
structure studies revealed alterations, such as fusion of the foot
processes of podocytes, and electron dense deposits in the cytoplasm

and basement membrane of these cells, which were considered as suggestive

7’8’9 In such studies, there have been

of immune complex nephritides.

only a few attempts to identify antigens that had stimulated the immune

globulins involved and to determine that these antigens were part of

10,11

immune complexes associated with lesions.
In studies of the pathogenesis of acute avian malaria, both anemia

aind nephritis of acute Plasmodium gallinaceum infections of chickens

vwere associated with the appearance of cold-active hemagglutinin for

106

trypsinized erythrocytes, and the concurrent presence of serum antigen
and antibody in the blood of the malarious chickens. Both anemia and
acute nephritis were induced in normal chickens by injection of plasma
which contained these immunologically active substances.12 In sUbsequent
work it was shown that both serum antigen and antibody could be eluted
from the washed blood cells of birds that had been injected with the
plasma of malarious chickens. Since these were the only antigens and
antibodies found reacting with the cells, it was suggested that immune
complexes involving serum antigen and its antibody were in part
responsible for the anemia of acute malaria of chickens.13

In the present studies the nephritides of acute avian malaria have
been investigated to determine whether or not immune complexes might
have been causal and to attempt to identify the antigens and antibodies
responsible. The results of these studies are presented in this

communication.
MATERIALS AND METHODS

Animals and Experimental Infections: The source and methods of main-

 

tenance of the white leghorn cockerel chickens, the P. gallinaceum
strain, and the B. rodhaini strain used for this work have been
described.12 Chickens for experimental studies were all infected by
intravenous inoculation of 108 or 106 parasitized erythrocytes, standard-
ized by methods described.14 Birds that furnished plasma for the
experiments were exsanguinated by cardiac bleeding under anesthesia as
described.12 Chickens that were to be subjected to postmortem study
were exsanguinated as described. Data on the parasitemia, expressed as
the percentage of parasitized erythrocytes (% PE) and anemia expressed

as red blood cell counts (RBC X 106), was obtained by described methods.12

107
Kidneys of experimental and control chickens were taken at autopsy.
Portions to be used for immunopathologic studies were taken from the
cortex area and cut into pieces 3-5 mm square which were quick frozen
in acetone-dry ice mixture and stored at -70 C until needed for study.
Kidney tissue from which antigen and antibody were to be extracted was

minced into very small pieces and processed immediately.

Reagent Antigens and Antibodies and Serologic Tests: Antigens for
these experiments consisted of serum antigen from the globulin of rats
with acute B. rodhaini infection (BrA), purified serum antigen (SA-l
and SA-2) from the plasma of malarious chickens, antigen leached from
blood cells (LA-3), and parasite antigen (PA) prepared as described.13
Antisera.were prepared against each of these antigens in chickens.1
Portions of the plasma from immunized chickens were precipitated and
the globulin conjugated with fluorescein isothiocyanate (FITC) as
described.13 Antibody to serum antigen from rats with acute B. rodhaini
infection was referred to as ABr, antibody to serum antigen from malar-
ious chickens as ASA, and antibody to parasite antigen as APA. FITC
conjugated anti-chicken 7S globulin was obtained from Nutritional
Biochemicals Corp., Cleveland, Ohio.

Double diffusion in gel and the tube bentonite flocculation tests
were used to detect and titer the antigens and antibodies present in

materials from malarious chickens following described methods.12’13

Study of Protein Secreted in Dropping§_of Chickens with Acute
P. gaZZinaceum Infection: Physical evidence of kidney disease was
sought for by studying droppings from malarious birds. The droppings,
which contained the urinary secretions, were Obtained at daily intervals

from infected and control chickens. Fecal material was in part avoided

108
by collecting the excrement that was predominantly white, or greenish,
from malarious birds. Approximately 10 gm of the droppings from experi-
mental and control chickens were each homogenized in a Waring blender
with 50 ml of veronal acetate buffer, pH 8.6, ionic strength of 0.1, for
1 minute at medium speed. The homogenate was then centrifuged at 800 g
for 5 minutes, the supernatant fluids recovered, and allowed to stand
overnight at 4 C. The supernatant was further clarified by centrifuga-
tion at 2000 g for 15 minutes at 4 C and each preparation was concentrated
to a volume of 4 ml by ultrafiltration at 4 C. These preparations were
stored at -18 C until needed for study. Because of discoloration and
turbidity, total protein concentrations were not determined on the
droppings extracts. They were tested for antigen and antibody in double
diffusion in gel tests, and subjected to immunoelectrophoresis and disc
electrophoresis study by methods described.13 The droppings extracts
were subjected to column chromatography with Sephadex G-100 following

described methods.13

Studies of Kidney Tissues of Chickens with Acute P._gaZZinaceum
Infection for Antigens and Antibodies: Kidney pieces taken from malarious
chickens at the peak of infection and from control birds were washed 4
times with cold phosphate buffered 0.85% NaCl saline (PBS) pH 7.2.
Approximately 5 volumes of cold PBS were added to the minced tissues
and the mixture homogenized in a Waring blender for 2 minutes at medium
speed. The homogenate was then centrifuged at 2000 g for 30 minutes at
4 C. The clear supernatant was collected and dehydrated to l/5th of
original volume by ultra filtration, or by dehydration in dialysis
tubing with polyethylene glycol treatment,at 4 C. This preparation

was tested for antigens.

109

The sediment of homogenized kidney tissue was resuspended and
washed with centrifugation in 5 volumes of PBS. After the 7th washing
the pellet was resuspended in 5 volumes of 0.1 N citric acid solution,
pH 2.5, and placed on a mechanical stirrer for 16 hours at 4 C. The
mixture was then centrifuged at 2000 g for 15 minutes at 4 C and the
supernatant recovered. This supernatant was neutralized to pH 7.0
with 0.1 N NaOH, dialyzed overnight against PBS at 4 C, and dehydrated
to a volume of 4.0 ml by polyethylene glycol treatment at 4 C. These
procedures were adapted from those described by Gallo15 and Houba et
al.16 This preparation was tested for antigen and antibody in double
diffusion in gel, and in the TBF tests. It was also subjected to study
after immunoelectrophoresis and disc electrophoresis as described by

Soni and Cox.13

Detection and Identification of Immune Complexes Associated with
Nephritis in Acute P. gallinaceum Infections of Chickens: Frozen kidney
tissue samples from malarious chickens, chickens that had been injected
with plasma of malarious chickens, and from control birds were oriented
in Lipshaw M-l embedding matrix and frozen to -20 C. Sections were cut
at 4 microns with a Lipshaw Cryotome, mounted onto microscope slides
precooled to -20 C, and fixed for 10 minutes in acetone. The slides
were then washed in PBS, pH 7.0, for 10 minutes and FITC conjugated
antiserum, diluted to 1:20 or 1:40, was added. The preparations were
incubated at 37 C for 30 minutes. They were then washed in PBS, pH 7.0,
for 10 minutes and a No. 1 cover glass was mounted with 90% glycerine in
PBS pH 7.0. The sections were examined with a Zeiss Fluorescope illumin-
ated with an Osram HBO 200 super high pressure mercury lamp using Excitor
filter II and a Barrier filter 50/44. Microphotographs were taken with

Kodak Tri-X-Pan film with an exposure time of 30 to 60 seconds.

110

EXPERIMENTS AND RESULTS

Antigen Extracted from the.Kidngy Tissues of Chickens with Acute
Ptggallinaceum Infection: When the material extracted from the homogene
ized kidney tissues of malarious chickens was tested in double diffusion
in gel tests with the plasma of recovered chickens, a single line of
precipitation was observed, indicating that there was at least one
antigenic component present in the extract (Figure 1A). Tests of the
extract with ABr indicated that it contained serum antigen. Tests with
APA.were negative. No reactions were seen in any of the tests with
material extracted from kidneys of normal chickens.

Immunoelectrophoresis reaction with antiserum to normal chicken
serum indicated that the serum proteins of the extract were IgM and
IgG (Figure 1B). In disc electrophoresis at least 23 protein bands
were observed in the extract from malarious chickens while none were
observed in tests of samples from control birds (Figure 1C). Stains

for carbohydrate, lipid, and DNA were negative.

Antibody Extracted from the Kidney Tissues of Chickens with Acute
P. gaZZinaceum Infection: The material extracted with citric acid
reacted in double diffusion in gel tests with serum antigens SA-l and
SA-2. A faint line of reaction was also seen with material leached from
cells of malarious blood (LA-3), but no reaction was observed in a test
with parasite antigen preparation (Figure 2A). The finding that antibody
to serum antigen, but not to parasite antigen, was present in the
extracted material was also confirmed with the tube bentonite floccula-
tion test using bentonite sensitized with parasite antigen and serum
antigen. Tests with parasite antigen were negative and antibody to serum

antigen was consistently detected.

111

The citric acid extracted material exhibited two lines of precipi-
tation in tests with anti-chicken globulin and anti-7S globulin, which
suggested that there was more than one class of globulin present (Figure
ZB and 2C). Material extracted from kidneys of normal chickens did not
exhibit these reactions.

Immunoelectrophoresis with anti-normal chicken globulin indicated
that the protein migrated in a manner similar to IgG, and that the
extract from normal kidney did not contain this protein (Figure 2E).

In disc electrophoresis the material exhibited 10 protein bands, indicat-
ing that protein other than IgG had been extravasated into the tissue.
Again, protein was not detected in the extracts of normal kidney

(Figure 2F).

To further substantiate that the material extracted from the kidneys
of malarious chickens was antibody, absorption was made of the extract
with equal amounts of SA-l, SA-2, and LA-3. The absorbed extract gave
no reactions when retested with the antigens to which it had previously

reacted (Figure 2G and 2H).

Study_of Extracts of Droppings of Chickens with Acute P. gaZZinaceum

 

Infection for Immunologic Activity: Extracts of droppings of malarious

 

chickens did not lend themselves to quantitative study of protein secre-
tion. However, in disc electrophoresis as many as 16 bands taking
protein stain were detected in the test of the extracts from malarious
birds while none were evident in tests of material from control chickens
(Figure 3D).

In immunoelectrophoresis with anti-chicken serum, reaction bands
suggestive of IgG and albumin also indicated that a diversity of serum

proteins were passing in the droppings (Figure 3C).

112

In double diffusion in gel tests the droppings extract reacted with
plasma of chickens with acute P. gaZZinaceum infection, A. Reaction with
ABr, B indicated that serum antigen was secreted (Figure 3A). Gel tests
for parasite antigen and antibody were negative. Tests with anti-7S
indicated that IgG had been secreted (Figure 38).

After molecular sieving through Sephadex G-100, protein bearing
fractions were collected as shown in Figure 4. Two peaks of concentra-
tion, one similar to globulin and the other to low molecular weight
protein, were consistently found in the extracts from malarious chicken
droppings. Antigen and antibody activities both were confined to the

globulin peak.

The Interrelationships of Serum Antigen.and Antibody in Plasma, and

 

in Droppings, to the Course of Acute P. ggllinaceum Infection: A group
of chickens were given a light infection of 106 parasitized erythrocytes.
Slides for estimating parasitemia, blood for RBC counts and droppings
for extraction were collected daily. Plasma for serologic tests was
collected at 2 day intervals. Both plasma and droppings extracts were
tested for serum antigen and antibody using the TBF test. Table 1 shows
the results of this experiment. Parasitemia was first detected on the
3rd day, reached a peak on the 6th, and fell rapidly thereafter.
Reductions in red cell counts were observed on the 2nd day and were
lowest on the 6th. Thereafter, the counts increased at about the rate
they had fallen and were normal again the 14th day. Serum antigen was
detected in plasma samples on the 2nd day but was not found in droppings
extracts until the 3rd, when both antigen and antibody were present.

The titers of serum antigen fell precipitously after the 8th day and

were no longer present in either plasma or droppings after the 10th.

113
Antibody was detected in both plasma and droppings from the 4th day

throughout the experiment.

Fluorescent Antibody¥Studies of Kidney Sections from Chickens with
Acute P._gaZZinaceum Infection: Sections of frozen kidney tissues from
malarious chickens were reacted with FITC conjugated ABr, ASA and APA.
The presence of extravasated immune globulin was detected with conjugated
A75. Examples of the reactions obtained are shown in Figure 5.

Immunofluorescent staining of kidney sections from malarious birds
was obtained with each of the conjugates. The staining with the ABr
and ASA conjugates showed diffuse "lumpy-bumpy" type of immunofluorescent
reactions. Similar types of reaction patterns were observed with the
A78 conjugate. Fluorescence with the APA conjugate was spotted, less
diffuse, and appeared to be confined to the capillary loop of the

glomerular tuft.

The Relationships of Parasitemia, Anemia, Serum Antiggn, and Anti—
body to Serum Antigen in Blood to Immunofluorescent Reactions of Kidney_
Tissues in Chickens Infected with P. gallinaceum: Parasitemia, red
blood cell counts, tests for serum antigen and antibody were made daily
on chickens brought to autopsy after P. gaZZinaceum infection. Frozen
kidney sections were tested with FITC conjugates of ABr, ASA, APA, and
A78, and evaluated as 0, 1+, 2+, 3+, or 4+ on the basis of intensity of
immunofluorescent activity. The results of the experiment are presented
(Table 2).

Parasitemia did not become evident until the 5th day of infection;
however, red blood cell counts declined slightly. Titers of SA and ABSA
were not detected in plasma until the 7th day; however, kidney sections

showed slight reactions with each of the conjugates on day 6 and were

114
near maximal on day 7. Thereafter each conjugate reacted strongly with
kidney sections until day 15, when reactions with ABr, ASA and APA.became
negative. These changes were accompanied by the disappearance of
parasites and SA from the blood. Blood titers of ABSA, and reactions of

the tissue sections with A7S,remained strong throughout the experiment.

Immunofluorescent Reactions of Kidneys of Normal Chickens after
ijection of Plasma from Chickens with Acute P. gallinaceum Malaria:
Immunofluorescent studies were made of the kidneys of normal chickens
that had been injected intravenously with plasma of malarious chickens
bearing serum antigen and antibody. Immunofluorescent reactions with
ABr, ASA and A73, which were similar to those seen in kidney sections
from malarious birds, were evident in chickens brought to autopsy at
24 and 48 hours after injection. Again the diffuse "lumpy-bumpy"
pattern of the fluorescence indicated that the reacting antigens and
globulins were in extravascular tissues. No reactions were obtained

in tests with APA conjugates in this experiment (Figure 6).

DISCUSSION

Earlier work indicated that there was an acute glomerulonephritis
associated with acute P. gallinaceum infections of chickens. The severity
of the nephritis was associated with the concurrent presence of serum
antigen and antibody in the blood of the malarious birds. When plasma
containing the antigen and antibody was injected intravenously into
normal chickens, nephritis similar to that seen in malarious chickens
was detected 24 hours after injection. Since these changes were not
produced by injection of chickens that had recovered from P. gaZZinaceum
infection, it was considered that they had been mediated by immunologic

mechanisms, rather than by blood permeability factors.12 In the present

115
work additional evidence was furnished indicating that kidney malfunction
was a part of the disease syndrome. Serum proteins were found in the
urinary part of droppings of malarious chickens soon after parasitemia
became patent and continued to be present at the time the birds were
well recovered from the parasitemia and anemia of acute infection.
Serum protein, which was not evident in extracts of normal chicken
kidneys, further indicated that extravasation of blood substances into
the kidney tissues had occurred.

Immunochemical studies of the protein found in the droppings indi-
cated that the larger portion was low molecular weight material. There
was, however, a substantial quantity of high molecular weight protein
that was eluted from the Sephadex column in the range of serum globulin.
Immunoelectrophoresis and disc electrophoresis studies of the extracts
confirmed the presence of globulin and albumin. In serologic tests,
the globulin bearing samples reacted with plasma of recovered chickens
which indicated the presence of antigen. Tests with plasma of acutely
malarious chickens indicated the presence of antibody. Samples bearing
low molecular weight protein failed to react. Further study of the
droppings extracts revealed that they reacted in gel with antibody to
serum antigen from rats with acute B. rodhaini infection. Reactions with
purified parasite antigen, or antibody to parasite antigen, were not
observed. Thus the only antigen and antibody detected in the urinary
secretions of the malarious chicken was the globulin associated serum
antigen and its antibody.

In an experiment, the relationships of parasitemia and anemia to
the presence of serum antigen and antibody in plasma and in urinary
wastes were studied. There appeared to be a relationship of the presence

of antibody to a rapid decline in red blood cell counts and the

116
extravasation of protein, as was indicated by the appearance of antigen
and antibody in the droppings. Serum antigen was detected in plasma
on the 2nd day when parasitemia was very low. It was first detected in
droppings on the 3rd day when both antigen and antibody were present.
From then until the 7th day, titers of antigen were higher than those of
antibody in both the plasma and droppings, suggesting that there was
an excess of the antigen. This was associated with the rapid fall in
red blood cell counts and increases in the titers of antigen and anti-
body secreted in the urine. From the 8th day antibody titers were
higher in both plasma and drOppings, and antigen was not detected after
the 10th day. This change was accompanied by a rapid improvement in
the red blood cell counts and a remarkable improvement in the appear-
ance of the chickens, in spite of the persistence of fairly high para-
sitemia. Changes in protein output in the urine were not evident since
the titers of antibody to serum antigen in the droppings extracts approxi-
mated those found in plasma.

Study of material extracted from the kidneys of malarious chickens
revealed the presence of serum proteins that were not present in
extracts of kidneys from normal chickens. Immunoelectrophoresis and
disc electrOphoresis again indicated that a variety of protein, ranging
from globulin to albumin, had been extravasated into the tissue. Sero-
logic tests of the extracts indicated the presence of globulin associated
serum antigen and its antibody. Tests with parasite antigen and its
antibody did not show reactions.

In the immunofluorescent study of kidney sections from chickens
with acute P. gallinaceum malaria, FITC conjugates of antibody to serum
antigen from rats with acute B. rodhaini infection, and antibody to

serum antigen from malarious chickens, both of which were prepared in

117
chickens, reacted strongly, giving a picture of diffuse granular type
of fluorescence which was described by Dixon17 as "lumpy-bumpy" and
associated with immune complex glomerulonephritis. Similar "lumpy-
bumpy" fluorescence was seen in sections reacted with conjugates of
anti-chicken 7S globulin. Sections from chickens with acute P. gaZZina-
ceum malaria also reacted with FITC conjugates of antibody to parasite
antigen that had been repared in chickens. In these tests the reacting
areas showed strong fluorescence, but diffuse immunofluorescence, as
was seen with antibody to serum antigen and with anti-7S, was not
evident. That is, it appeared that the parasite antigen had not dif-
fused into the tissues or the glomerular tuft as extensively as had
serum antigen.

Sections from kidneys of uninfected chickens with nephritis induced
by injections of plasma from malarious chickens also reacted with conju-
gates of antibodies to serum antigens of babesial and plasmodial origin,
and with anti-7S conjugates, just as described above, except that the
diffuseness of immunofluorescence was less than that observed in sections
from acutely malarious chickens. Tests with conjugates of antibody to
parasite antigen did not give immunofluorescent reactions with sections
taken from the kidneys of these chickens.

In an experiment showing the relationships of parasitemia, anemia,
serum antigen and antibody in blood, to immunofluorescent activity in
kidney sections prepared from chickens sacrificed daily after infection,
the sections were tested with conjugates of antibodies to serum antigens
of babesial and malarious origins, anti-parasite antigen and with anti-
chicken 78 globulin. The beginning of immunofluorescent activity with
each of the conjugates was associated with the concurrent presence of

serum antigen and antibody in blood of the chickens. Intensity of the

118
reactions, graded from zero to 4 plus, was approximately equal for each
conjugate from the 6th through the 13th day. Reactivity with the anti-
bodies to the serum antigens and parasite antigen all ceased on the same
day. The reversion of these reactions to negative tests was associated
with the disappearance of serum antigen from the blood and the recovery
from parasitemia. Reaction with tbe anti-7S conjugate continued to be
strong for the remaining observations of the experiment, and were associ-
ated with the presence of antibody to serum antigen in the blood of the
animals. Thus it was indicated that serum globulins were extravasated
into the kidney tissues for some time after recovery from acute malaria,
just as was indicated by the presence of antibody to serum antigen in
the droppings of recovered chickens.

In supposing that immune complex nephritis would be dependent upon
the presence of both soluble antigen and antibody in the blood, and that
antigen excesses are usually associated with the disease, it seems that
the relationships of the globulin associated serum antigen and its anti-
body in the blood, in the kidney tissues, and in the urinary wastes of
the malarious birds fulfill these requirements.

In the study of the antigens present in the blood of chickens with
acute P. gallinaceum malaria, there was but little evidence that soluble
parasite antigen was present free in the plasma, even though a high
titer of antibody to parasite antigen was evident, along with a high
titer of antibody to serum antigen. On the other hand, a high titer
of serum antigen, which was clearly shown to be serologically unrelated
to parasite antigen, was a constant finding.13 Thus the soluble serum
antigen, and its antibody, were the obviously available ingredients in

the plasma for forming immune complexes in these experiments.

119

The reactions of antibody to parasite antigen with the kidney
sections from malarious chickens must bear the burden of being the best
evidence found that parasite antigen and its antibody had a role in the
nephritis of acute P. gaZZinaceum malaria of chickens. Since this
antigen and antibody were not evident in the nephritis induced in
normal chickens by injections of malarious plasma, it was further indi-
cated that serum antigen and its antibody were important mediators of
.nephritis in these experiments.

The results of these experiments lead to the suggestion that com-
plexes of a globulin associated serum antigen which was serologically
unrelated to P. gaZZinaceum parasite antigen, and which is found in the
blood of animals with acute red blood cell infections of parasites other
than those of the genus Plasmodium, was along with its antibody in
part the mediator in glomerulonephritis in chickens with acute P. galli-
naceum malaria. However, it is not implied that the immune complexes
alone were the causal agents. It is probable that the 81C (C3) fraction
of complement, activated by the complex, had a major role in altering
cell membranes to allow cellular hydration and in altered vascular
permeability as was suggested by Ward and Conran.10’11

The results of these experiments do not indicate that serum anti-
gen and antibody are the sole mediators of nephritis in acute malaria.
Glomerulonephritis was found to be associated with acute Plasmodium
chabaudi malaria of rats, where it was conclusively shown that serum
antigen was not elaborated during acute infection. However, in these
rats it was indicated that soluble complexes of parasite specific
antigen, and its antibody, were present in blood, in the kidney tissues,
and were secreted in the urine.18 Thus, while the antigens and anti-

bodies involved differed in the two infections, the mechanisms implicated

120
in acute P. chabaudi infection of rats, and acute P. gaZZinaceum infec-
tion of chickens, seem essentially the same. That is, both diseases
appear to have been mediated by immune complexes.

The role of cold—active hemagglutinin for trypsinized erythrocytes
in acute glomerulnephritis has not been evaluated. In a report of
nephritis associated with acute B. rodhaini infections of rats, it was
suggested that the hemagglutinin might have been in part responsible,
since the titers were correlated with the severity of kidney damage.19
This agglutinin was also found in acute P. gaZZinaceum infections of

12

chickens. The role of the agglutinin in anemia has been studied and

will be discussed in a subsequent communication.

REFERENCES

1. Bright, R. 1827. Cited by Carpenter, C. B. 1970. Immunological
aspects of renal disease. Ann. Rev. Med. 21:1-16.

2. Maegraith, B. G., and Findlay, G. M. 1944. Oliguria in black
water fever. Lancet 2:403—404.

3. Spitz, S. 1946. The pathology of acute falciparwn malaria.
Milit. Surg. 99:555.

4h Maegraith, B. G. 1948. Pathological processes in malaria and
black water fever. Blackwell Scientific Publications, Oxford,
England.

5. Kibuko-Musoke, J. W., and Butt, M. S. R. 1967. Histological fea-
tures of the nephritic syndrome associated with quartan malaria.
J. Clin. Path. 20:117-123.

6. Dixon, F. J. 1968. The pathogenesis of glomerulonephritis. Am.

J. Med. 44:493-498.

L0-

1.1.-

12!.

13u

14.

15.

121

Allison, A. C., Hendrickse, R. G., Edington, G. M., Houba, V.,
DePetris, S., and Adeniyi, A. 1969. Immune complexes in the
nephritic syndrome of African children. Lancet 1:1232-1238.
Hendrickse, R. G., Adeniyi, A., Edington, G. M., Glasgow, E. F.,
White, R. H. R., and Houba, V. 1972. Quartan malarial nephrotic
syndrome: Collaborative clinico-pathological studies in Nigerian
children. Lancet 1:1143-1149.

Boonpucknavig, V., Boonpucknavig, S., and Bhanarapravati, N.

1973. Plasmodium berghei infection in mice: An ultra structure
study of immune complex nephritis. Am. J. Path. 70:89-108.

ward, P. A., and Conran, P. B. 1966. ImmunOpathologic studies of
simian malaria. Mil. Med. (Suppl.) 131:1225-1232.

Ward, P. A., and Conran, P. B. 1969. Immunopathology of renal
complications in simian malaria and human quartan malaria. Mil.
lied. (Suppl.) 134:1228—1236.

Soni, J. L., and Cox, H. W. l973a. Pathogenesis of acute avian
Inalaria. I. Immunologic reactions associated with anemia and
Iiephritis. In preparation.

ESoni, J. L., and Cox, H. W. l973b. Pathogenesis of acute avian
nmalaria. II. Study of antigens and antibodies associated.with
anemia of acute Plasmodium gaZZinaceum infection of chickens.

III preparation.
Corwin, R. M., and Cox, H. W. 1969. The immunologic activity
(Df' the nonspecific serum antigens of acute hemosporidium infection.
Mil. Med. (Suppl.) 134:1258-1265.

Calla), G. R. 1970. Elution studies in kidneys with linear deposi-

tiotl of immunoglobulins in glomeruli. Am. J. Path. 61:377-385.

165-

17'-

153.

19-

122
Houba, V., Allison, A. C., Adeniyi, A., and Houba, J. E. 1971.
Immunoglobulin classes and complement in biopsies of Nigerian
children with the nephrotic syndrome. Clin. Exp. Immunol. 8:
761-774.
Dixon, F. J. 1972. Glomerulonephritis and immunopathology. .23
Immunobiology, by Good, R. A., and Fisher, D. W., Chapter 17, p.
167-173. Sinauer Associates, Inc., Publisher, Stamford, Conn.
Musoke, A. J. 1973. Immunologic and pathologic studies of
infections with rat adapted Plasmodium chabaudi. M.S. Thesis,
Michigan State University, East Lansing, Michigan.

Iturri, G. M., and Cox, H. W. 1969. Glomerulonephritis associa-

ted with hemosporidian infection. Mil. Med. (Suppl.) 134:1119-1128.

 

L Ii! III“

 

123
TABLE 1
The average percent of parasitized erythrocytes (PE), the red blood cell
COlmtS (RBC X 106), the average of the titers of serum antigen (SA) and
antibody to serum antigen (ABSA) in plasma, and fecal droppings from

chickens with acute Plasmodiwn gallinaceum malaria.

 

Days Post RBC 6 Plasma Plasma Fecal Fecal
Infection P.E. X 10 SA ABSA SA ABSA

l 0 2.67 0 0 0 0

2 + 2. 15 32 0 0 0

3 3. 66 2.02 N* N* 32 12

4 25.9 1.49 192 48 64 16

5 42 0 .94 N* N* 96 20

6 81. 2 0.58 288 178 96 20

7 47 0. 78 N* N* 96 24

8 22 1.39 298.6 512 96 136

9 16.5 1. 61 N* N* 80 192

10 13 1.90 20 288 16 256

ll 3 2. 37 N* N* 0 128

12 2 2. 42 0 160 0 128

13 1 2.65 N* N* o 128

14 - 2. 79 0 48 O 32

 

*
N0 test. Plasma samples were taken on alternate days.

124
TABLE 2

Paras itemia (av. ‘7. PE) and anemia (av. RBC X 106) in chickens infected
with Plasmodium gaZZinacewn, the titer of serum antigen (SA) and anti-
body to SA (ABSA) in plasma, the intensity of fluorescent activity in
fluorescent antibody tests (FAT) with conjugated antibody to serum
antigen from rats with acute Babesia rodhaini infection (ABr), antibody
to serum antigen from malarious chickens (ASA), antibody to P. gaZZina-
cewn parasite antigen (APA), and anti-chicken 7S globulin, in frozen
kidney sections from a chicken brought to autopsy at daily intervals

af ter infection.

 

 

 

Days Post Av. Av. RBC Results of FAT
Infection Z P.E. 106 SA ABSA ABr ASA APA A78
1 0 3.02 0 0 0 0 0 0
2 0 2.73 0 0 0 0 0 0
.3 0 2.72 NT* NT 0 0 0 0
4. +** 2.57 0 0 0 0 0 0
.5 0.31 2.55 0 0 0 0 0 0
cs 2.3 2.33 0 0 + + + +
7 12.2 2.26 128 128 3 3 3 2
8 27.7 2.20 512 256 4 4 4 3
9 24.5 2.01 NT NT NT NT NT NT
10 28.9 1.32 1024 512 4 4 4 4
11- 28.4 1.16 512 128 3 3 3 2
12 18.4 1.01 64 256 2 2 2 3
13 7.2 1.13 32 256 2 2 2 3

14 3 .0 1.25 NT NT NT NT NT NT

125

TABLE 2 (CONT'D.)

 

 

 

 

 

Days Post Av. Av. RBC Results of FAT
Infection 2 P.E. 106 SA ABSA ABr ASA APA A73
15 0.75 1.33 0 128 0 0 0 2
16 + 1.35 NT NT NT NT NT NT
17 + 1.70 0 64 0 0 0 2
18 O 1.95 NT NT NT NT NT NT
19 0 1.69 O 512 0 0 0 4
20 0 NT NT NT NT NT NT NT
21 0 1.85 0 256 0 0 0 3
3‘:
Not tested.
*3:

Parasites detected, too few to count.

 

 

 

126

Figure 1. Reactions of antigen extracted from the kidneys (KA) of
chickens with acute Plasmodium gallinaceum infection.

l-A. Reaction in double diffusion in gel test with plasma of
chickens recovered from P. gaZZinaceum malaria.

l-B. Reaction of KA, and normal control, after immunoelectrophore-
sis with anti-normal chicken serum (Top), compared to the immunoelectro-
phorogram of normal chicken serum (Bottom).

l-C. Disc electrophoresis of KA (Left) compared to normal chicken
globulin (Center) and extract of normal kidney (Right) after staining

for protein.

127

   

Figure l

 

 

 

128

Figure 2. Tests of antibody extracted from the kidneys (KAb) of
chickens with acute Plasmodium gaZZinaceum infection.

2-A. Reactions of KAb in double diffusion in gel tests showing
positive reactions with serum antigen from malarious chickens (8A1 and
SA2) but no reactions with parasite antigen (PA). A faint precipitin
line not seen in the photograph was seen in the test with material
leached from the cells of malarious blood (LA3).

2-B. Reaction of KAb with anti-normal chicken globulin (ACG). No
reaction was seen with extract from normal kidney (N).

2-C. Reaction of KAb with anti-chicken 7S globulin and absence of
reaction with N.

2-D. Reaction of KAb with plasma of recovered chickens (R) indi-
cating that antigen was associated with KAb.

2-E. Immunoelectrophoresis with anti-chicken globulin of KAb (Top)
and extract of normal kidneys (Bottom) compared to whole chicken globulin.

2-F. Disc electrophoresis of KAb compared to extract of normal
kidney after staining for protein.

2-G. Reaction of KAb with SAl, SA2, and LA3 before absorption.

A reaction, not seen in the photograph, was observed with LA3 in the
original slide.

2-H. Absence of reaction with SAl, SA2, and LA3 of the absorbed

supernatant (AS) from KAb, after incubation with SAl, SA2, and LA3.

 

Figure 2

 

 

 

130

Figure 3. Reactions of extracts of droppings from chickens with
Plasmodium gaZZinaceum malaria.

3-A. Reaction of extracts of droppings on day 4 with antibody to
serum antigen from rats with acute Babesia rodhaini infection (B) and
below with plasma of malarious chickens (A) compared to control extract
(N).

3-B. Reactions of droppings extract from day 8 with anti-chicken
7S globulin (A7S) compared to N.

3—C. Immunoelectrophoresis with anti-chicken globulin of droppings
extracts from malarious chickens (Top) and extracts from droppings of
normal chickens (Bottom), compared to normal chicken globulin.

3-D. Disc electrOphoresis of droppings extracts from malarious

chickens (Left) and normal chickens (Right) after staining for protein.

 

_- .__—a. .,.

o--- -—

131

C

Figure 3

"f
a.

!.' '

 

 

 

132
Figure 4. Elution profile of protein eluted from extracts of drop-
pings from chickens with acute Plasmodium gaZZinaceum malaria, after
column chromatography with Sephadex G-100. Column size: 2.5 x 100 cm.
Packed column: 2.5 x 90 cm. Charge: 4 m1 of extract. Elution with:
Borate buffered saline, pH 8.2, I = 0.16. Flow rate: 15 ml/hour.

Fraction volume: 3 m1.

133

CON on.

 

a unawfim

«.02 must mgr—.041“. £3.50
0! ON. 00. on

on F.

 

 

3
um 082 IV A1ISN30 'Ivoudo

 

 

 

134
Figure 5. Reactions of kidney sections from chickens with acute
Plasmodium gaZZinaceum malaria, with fluorescein isothiocyanate conju-
gated antibody to serum antigen from rats with acute Babesia rodhaini
infection (A), antibody to serum antigen from chickens with acute P.
gallinaceum malaria (B), antibody to P. gaZZinaceum parasite antigen (C),

and anti-chicken 7S globulin (D) (400 X).

   

Figure 5

 

{I

136
Figure 6. The reactions of kidney sections from chickens taken 48
hours after injection of plasma from chickens with acute Plasmodium
gaZZinaceum malaria, with fluorescein isothiocyanate conjugated antibody
to serum antigen from rats with acute Babesia rodhaini infection (A),
antibody to serum antigen from chickens with acute P. gaZZinaceum
malaria (B), anti-chicken 7S globulin (C), and antibody to P. gaZZina-

ceum parasite antigen (D) (400 X).

 

 

 

Article 4

PATHOGENESIS OF ACUTE AVIAN MALARIA

IV. ANEMIA MEDIATED BY THE COLD-ACTIVE AGGLUTININ FOR TRYPSINIZED

ERYTHROCYTES FROM THE BLOOD OF CHICKENS WITH ACUTE

PLASMODIUM GALLINACEUM INFECTION

 

Jiya L. Soni and Herbert W. Cox
Department of Microbiology and Public Health,
Michigan State University

East Lansing, Michigan 48823

(To be submitted to Infection and Immunity)

 

PATHOGENESIS OF ACUTE AVIAN MALARIA
IV. ANEMIA MEDIATED BY THE COLD-ACTIVE AGGLUTININ FOR TRYPSINIZED
ERYTHROCYTES FROM THE BLOOD OF CHICKENS WITH ACUTE

PLASMODIMM GALLINACEMM INFECTION

*
Jiya L. Soni and Herbert W. Cox

Department of Microbiology and Public Health,
Michigan State University,

East Lansing, Michigan 48823

*

U.S. State Department, Agency for International Development,
Fellow. Present address: College of Veterinary Science and Animal
Husbandry, Jawaharlal Nehru Agricultural University, Jabalpur,

Madhaya Pradesh, India.

138

 

139
ACKNOWLEDGEMENTS
Research here reported is from a thesis entitled, "Pathogenesis of

Acute Avian Malaria,"

submitted by the principal author, in partial
fulfillment of the requirements for the Doctor of Philosophy degree at
Michigan State University. Participation in this program was made
possible by a Fellowship from the U.S. State Department Agency for
International Development.

This research was supported in part with funds from Grant No.
AI-08508 from the National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland, and with support

from the Michigan Agricultural Experiment Station. This publication is

Journal Article from the Michigan Agricultural Experiment Station.

ABSTRACT

A cold-active agglutinin was absorbed from the plasma of chickens
with acute PZasmodEum gallinaceum malaria with trypsinized type "0"
human erythrocytes. It was first detected at the beginning of parasi-
temia, reached its highest titer just prior to the parasitemia-anemia
crises, and thereafter was detected only at low titer. Reinfection of
the chickens did not produce a marked elevation of the titers. The
agglutinin was found to be associated with the 19S and 7S globulin
fractions of malarious chicken blood, but cleavage with 2-mercapto-
ethanol indicated that it was primarily of the IgM class of antibody.
In serologic tests the agglutinin reacted only with trypsinized erythro-
cytes, anti-chicken globulin and with anti-chicken 7S globulin. It did
not react with any of the antigens or antibodies detected in the blood
ofInalarious chickens. Neither were titers of antigen and antibody in
malarious plasma other than the agglutinin altered by the absorption.

When the absorbed agglutinin was injected intravenously into normal

 

140
chickens it produced an anaphylactic-like shock and caused a 25% reduc-
tion in red blood cell counts within 48 hours. Plasma samples collected
during this interval showed signs of hemolysis. Immunofluorescent
study of blood cells removed from the injected birds showed reactions
with conjugated anti-chicken globulin, but did not show reactions with
conjugates of antibody to any of the antigens found in blood of malarious
chickens. The absence of fluorescent activity 3 days after injection
suggested that erythrocytes that had reacted with the agglutinin had
been removed from the circulation. Anemia following the injection of
malarious plasma that had been absorbed free of agglutinin indicated that
anemia inducing factors other than the agglutinin were also present.
While the antigenic determinants for the agglutinin were not indicated,
the data suggest that the agglutinin is an autoantibody, and clearly

indicates that it may have a causal role in malarial anemia.

INTRODUCTION

Anemia that was incommensurate with parasitemia has come to be a
well known part of disease in red blood cell infections. The associa-
tion of a positive Coomb's test with anemia of Plasmodium berghei infec-
tions of rats led to the suggestion that the anemia might have been
mediated by autoimmune substances (19). Similar suggestions were made
for the anemia associated with acute Plasmodium Zophurae infections of
ducklings and for Anaplasma marginale infections of cattle (9,10,13).

The association of cold-active agglutinin with anemia, splenomegaly,
and erythrophagocytosis in A. marginale, P. berghei and Babesia
Podhaini infections led to the suggestions that in each of these cases
anemia may have been in part due to this autoantibody-like substance

(3,14,15). Barrett at a2. (1) demonstrated cold-active agglutinin

 

141
sassociated with anemia of Plasmodium Zophurae infection in chickens
Insing trypsinized human type "0" erythrocytes. They associated the
sagglutinin with 198 globulin and found its activity to be destroyed
15y 2-mercaptoethanol treatment. The titers of the agglutinin did not
cdiminish after recovery from malaria and subsequent injections of
(erythrocyte from normal chickens resulted in a decrease in the titers.

The association of cold-active agglutinin for trypsinized human
"0" erythrocytes with acute malaria was confirmed using acute Plas-
rnodium gaZZinaceum infections of chickens. However, it was found
that the agglutinin titers tended to fall quickly after recovery from
parasitemia (16). Further, questions concerning the persistence of
high titers of agglutinin were raised since it was found that P.
Zophurae at this laboratory and at four others was contaminated with
an anemia inducing virus agent which caused a disease in ducks that
mimicked acute malaria (7,8). The virus of duck infectious anemia
(DIA) could be transmitted as aerosol from infected to clean birds,
including chickens (8). Before starting the present work our strain
of P. gallinaceum was extensively screened for DIA virus, all work:.
with DIA was discontinued, and our animal rooms were sanitized.

Anemia of acute P. gaZZinaceum infection in chickens was found to
be due in part to immune complexes of soluble antigens and antibodies
found in the blood of the birds during acute infection (17). The role
of the cold-active agglutinin has also been studied. The results of

the studies are reported in the present communication.
MATERIALS AND METHODS

Experimental animals and infections. Mature White Leghorn cockerels

for the experiments were obtained from Rainbow Trails Hatchery, St.

 

142
ZLouis, Michigan, as day-old chicks and reared in departmental animal
:facilities. The P. gaZZinaceum infection was obtained from Dr. Julius
1?. Kreier, Department of Microbiology, Ohio State University, Columbus,
(Ohio. After extensive testing for DIA virus contamination, it was
sstored in liquid nitrogen, or maintained by blood passage in chickens.
IFor this research it was considered essential that the parasites be
Inaintained at maximal virulence. Therefore severity of anemia and
‘moribund condition of the birds were the criteria used in selecting
infected birds to be used for passage. The methods of inoculation,

evaluating anemia, and parasitemia have been described (16).

Detection of cold-active agglutinin for trypsinized egythrocytes.
The agglutinin was detected and titrated in plasma from malarious
chickens using trypsinized human type "0" erythrocytes prepared by
methods modified from those of Cox et a1. (3) and have been

described (16).

Absorption and elution of agglutinin. Chickens infected with 108

parasitized erythrocytes that had been washed with 0.78% NaCl solution
were exsanguinated at the peak of parasitemia and anemia, and the blood
was added 10 parts to 1 part heparinized saline (100 I.U. Sodium
heparin/ml of 0.78% NaCl solution). After centrifugation at 800 g for

15 minutes, the plasma was recovered and stored at -18 C until tested.
For the test frozen plasma was thawed and clarified by centrifugation

at 2000 g for 30 minutes at 4 C and 10 m1 of plasma was added to 5 m1

of packed trypsinized human type "0" cells. After mixing, the suspension
was held at 4 C for 4 hours. The plasma was removed after centrifugation
and the cells were washed 4 times with cold 0.85% NaCl solution. The

packed cells were then suspended in an equal volume of the salt solution

 

143
and incubated at 37 C for 30 minutes. After centrifugation at 1500 g
for 10 minutes, the supernatant was recovered, placed in dialysis
tubing and dehydrated to l/5th volume by polyethylene glycol treatment
at 4 C. This material was then tested for agglutinin with trypsinized

human type "0" erythrocytes, and was stored at -18 C.

In vivo tests of cold-active agglutinin for biological activity,
Four chickens were injected intravenously with the agglutinin that had
been standardized to contain 4.0 mg of protein per inoculum. Four
other chickens were inoculated with eluates prepared from trypsinized
human "0" red cells used to absorb plasma of normal chickens. The
absorbed plasma was tested for anemia inducing factors by injection of
4 m1 intravenously into each of 4 other normal chickens. Anemia follow-
ing the injections was determined by daily red blood cell counts over a
10 day period as described (16).

Slides from the blood of chickens injected with agglutinin.were
prepared and tested for reactivity with fluorescein isothiocyanate
(FITC) conjugates of anti-chicken whole globulin, anti-serum antigen
from rats with acute B. rodhaini infection (ABr), and anti-P. gaZZinaceum

parasite antigen (APA) prepared as described (17).

Immungchemical'prpperties of cold-active agglutinin. Four m1

 

(160 mg) of globulin salted from the plasma of malarious chickens at
4 C with 50% saturated ammonium sulphate solution was subjected to study
by column chromatography using Sephadex G-200 as described (17). Each
sample from the column was tested for agglutinin activity with trypsin-
ized human type "0" cells as described (16).

The agglutinin was subjected to 0.1 M 2-mercaptoethanol (2-ME)

treatment as described by Chan and Deutsch (2). Reductive cleavage

 

144
was determined by testing the treated material with trypsinized "O"
erythrocytes.

Absorbed agglutinin and 2-ME cleaved agglutinin were studied by
immunoelectrophoresis and reacted with anti-normal chicken globulin
following described methods (17). Disc electrophoresis study of the
agglutinin was made following described methods (17).

The agglutinin, the 2-ME cleaved agglutinin and the absorbed
plasma were each tested for reactivity with serum antigen and antibody
to serum antigen of rat babesiosis origin, using the tube bentonite
flocculation (TBF) test as described by Thoongsuwan and Cox (l8) and
Soni and Cox (16). The agglutinin was also tested in double diffusion
in gel tests following described methods (6,17). Reactions were tested
for with: anti-normal chicken globulin, anti-chicken 7S globulin,
plasma of chickens recovered from P. gallinaceum infection, plasma of
chickens with acute P. gaZZinaceum infection, anti-P. gaZZinaceum
parasite antigen, P. gaZZinaceum parasite antigen, and purified serum

antigen from chickens with acute P. gaZZinaceum infection.
RESULTS

Absorption and recovery of cold-active agglutinin frompplasma of
chickens with acute P. gallinaceum infection. The titer of cold-active
agglutinin for trypsinized human type "0" erythrocytes, before and
after absorption, in the plasma from 5 malarious chickens, and the titer
of the agglutinin eluted from the cells, are shown in Table 1. The
average agglutinin titer of 122 for the plasma was reduced to zero after
absorption. The material eluted from the cells had a titer of 51. The
average titer for the 4 chickens hyperimmunized by repeated P. gaZZina-

ceum infection was 64. Cleavage with 2-ME destroyed agglutinin activity

 

145
in plasma from malarious chickens, as well as the low titers of agglutinin
found in plasma of normal chickens and the residual titers found in
plasma of birds that had been hyperimmunized by repeated P. gallinaceum

infection.

In vivo effects of cold-active agglutinin on normal chickens. The
red blood cell counts on chickens injected intravenously with eluted
agglutinin are shown in Table 2. Injection of the agglutinins produced
a drop of 14.6% in the number of circulating erythrocytes within 24 hours.

The maximum reduction of nearly 25% was attained on day 2. Recovery

 

from blood loss was evident after the 7th day. Immediately after injec-
tion of the agglutinin the recipient chickens went into anaphylactic-
like shock. Breathing was labored, their combs became cyanotic and the
birds were near collapse. About 2 hours were required before they
recovered. Plasma taken at 24 and 48 hours after injection showed dis-
tinct hemolysis.

Injection of the absorbed plasma produced a drop of 30% in the red
cell counts 3 days after injection (Table 3). Blood cell counts did not

revert to normal levels until 9 days after injection.

Immunochemical studies of the cold-active agglutinin. Protein

 

concentration and the titers of the agglutinin in fractions from globulin
salted from malarious chickens with‘50% saturated ammonium sulphate
solution after Sephadex G-200 chromatography are shown (Figure 1). Pro-
tein bearing column sample numbers 53 through 76 were positive when
tested with trypsinized cells, the highest titers being in samples 59
through 65. The remainder of the samples did not exhibit agglutinin
activity. The elution pattern indicated that agglutinin was located

primarily in the 195 peak but was also present in 78 fractions.

146

In double diffusion in gel tests the cold-active agglutinin (CA)
reacted with anti-chicken whole globulin (ACG) and with anti-chicken
7S globulin (A73) (Figure 2-1). Tests with anti-P. gaZZinaceum para-
site antigen, parasite antigen, purified serum antigen from malarious
chickens and antibody to serum antigen, were all negative.

In immunoelectrophoresis of column fraction pools, pool 1, consist-
ing of fractions 50-61, gave a precipitin line consistent with pure l9S
globulin while pool 2, consisting of fractions 62-70, showed a mixture
of 19S and 7S (Figure 2-2).

The reactions of absorbed agglutinin before and after 2-ME cleav-
age with anti-chicken globulin in immunoelectrophoresis are shown in
Figure 2-3. In the test shown (1) the agglutinin (top well) shows the
presence of both 193 and 7S globulin. The reaction of material absorbed
from normal plasma (N) showed similar but less distinct lines. The 2-ME
cleaved agglutinin (ME) in the lower well did not show any of the lines
seen with the uncleaved agglutinin (top well).

In disc electrophoresis the agglutinin exhibited 7 bands that took
protein stain. A preponderance of 19S globulin was indicated by the
presence of a heavy band taking protein stain which remained primarily

in the spacer gel (Figure 2-4).

Reactions of erythrocytes of chickens injected with cold-active

 

agglutinin with fluorescein isothiocyanate (FITC) conjugated antibodies.

 

Slides prepared from the blood of chickens after intravenous injection
of cold-active agglutinin were tested with FITC conjugated antibodies
to antigenic substances found in the blood of chickens with acute P.
gaZZinaceum infection, with conjugate of anti-chicken whole globulin,

‘and with anti-chicken 7S globulin. Immunofluorescent activity with

 

147
anti-whole globulin was observed about the periphery of erythrocytes
taken from the injected birds 24 hours after injection. The number of
cells showing fluorescent activity was substantially reduced at 48
hours and was no longer detected after 3 days. Blood from chickens
injected.with control material showed no fluorescent activity. FITC
conjugated antibody to serum antigen and to parasite antigen did not
show fluorescence (Fig. 2-5).

Results of tests for agglutinin, serum antigen and antibody to
serum antigen in plasma of malarious chickens, before and after absorp-
tion, are presented (Table 4). Absorption with trypsinized cells
removed all of the agglutinin from the plasma without affecting the
titers of serum antigen and antibody.' Agglutinin but no serum antigen
or antibody was detected in the eluate from the cells used for

absorption.

.DISCUSSION

These experiments have confirmed observations of Cox et al. (3)
that the anemia of acute malarial infections is associated with the
presence of cold-active agglutinin for trypsinized erythrocytes. They
also confirm the observations of Barrett et al. (1) and Soni and Cox
(16) that this, or a similar, agglutinin can be detected and titrated
in the blood of chickens with acute malaria using human type "0" tryp-
sinized'erythrocytes.

The observation of Barrett et a1. (1) that the agglutinin detected
with trypsinized cells, after Sephadex G-200 column chromatography of
globulin of malarious chickens, was exclusively l9S globulin was not
confirmed. A portion of the protein eluted from our column in the area

of 7S globulin contained agglutinin activity. Our immunoelectrophoresis

 

148
study also indicated the presence of 7S globulin in the eluted material.
Neither did we find that reinoculation of chickens recovered from acute
malaria with infected erythrocytes greatly increased the titer of the
agglutinin as was reported by Barrett et al. (1).

While it is possible that P. Zophurae infections of chickens differ
from those of P. gaZZinaceum, it is pointed out that P. Zophurae used in
the past at this laboratory,‘and at others, was contaminated with DIA
virus and that acute DIA closely mimicks acute malarial infection, even
to the extent that ducks recovered from acute DIA were resistant to

plasmodial challenge (7,8). In unpublished work from this laboratory

 

it was found that agglutinin to trypsinized erythrocytes appeared in

the blood of ducks with acute DIA and that the titers of the agglutinin

persisted long after apparent recovery. We do not doubt that the

agglutinin.was raised during acute P. Zophurae infection, but a possible

role of acute and chronic DIA virus infection must be considered in

attempts to draw conclusions about experiments performed with P. Zophurae.
It was found that intravenous injections of the agglutinin pro-

duced anemia in normal chickens. Red blood cell counts of the experi-

mental birds were reduced by nearly 15% over those of controls after

24 hours and to nearly 25% on day 2. Recovery from the anemia.was not

complete until the 9th day after injection. The birds injected with

the agglutinin also suffered an anaphylactic-like shock characterized

by extreme prostration, blanched or cyanotic boms, and labored breath.

However, none of the birds died and most had recovered after 2 hours.

That the shock could have been related to the agglutinin was further

indicated by hemolysis of all plasma samples taken from these birds on

days 1 and 2. No signs of shock were seen in the chickens injected with

control material and all plasma samples were clear.

149

Fluorescent antibody studies of blood slides obtained from the
chickens using a FITC conjugate of anti-chicken globulin indicated that
the agglutinin had reacted with the erythrocytes of the recipient
chickens. The marked reduction in immunofluorescent activity after the
3rd day suggested that the reacted cells had been removed from the
circulation.

Destruction of the hemagglutinating activity after treatment with
2-ME indicated that the agglutinin itself was a 193 immune globulin
as suggested by Barrett et al. (1). It is therefore probable that in
column chromatography study the hemagglutination activity present in
samples eluted from the column along with the 7S gldbulin was 198
contaminant. This information, along with the fact that it was readily
dissociated from the trypsinized cells, suggests that the agglutinin
was in fact cold-active IgM.

Aside from the serologic and in vivo reactions with erythrocytes
and with anti-chicken globulin, the agglutinin did not react in serologic
tests with any of the antigens or antibodies found to be associated
with P. gaZZinaceum infections of chickens. Further, the blood cells
from chickens injected with agglutinin did not react with FITC conju-
gates of any of the antibodies prepared against antigens found in the
blood of malarious chickens. The cells reacted only with anti-chicken
globulin conjugate.

The information from these experiments indicates this agglutinin
is antibody to red blood cell substances that are common to erythro-
cytes of heterologous species of animals, and that it has a role in the
pathogenesis of acute P. gallinaceum infections of chickens. While the
antigenic determinants for the agglutinin have not been indicated, the
present work furnishes further indirect evidence that it might be an

autoantibody as was suggested by Cox et al. (3).

 

150

Plasma that had-been'absorbed free of agglutinin still contained
anemia inducing substances. Such plasma when injected into normal
chickens produced a 30% reduction in red blood cell counts within 3
days. This substantiates the evidence that immune complexes of serum
antigen and antibody might have a role in anemia (17).

Dacie (5) has pointed out that the association of cold-active
agglutinin for trypsinized erythrocytes with congenital and idiopathic
anemias in man is a common finding and that the agglutinin is generally
considered to be an autoantibody that is causal in anemia. This, or a
similar type of agglutinin, has now been associated with anemia in a
diversity of animals infected with agents of equal diversity. Anemia
associated with the agglutinins was found in acute anaplasmosis of
cattle, acute B. rodhaini infection of rats, P. berghei infection of
rats, P. chabaudi infection of rats and mice, acute equine infectious
anemia virus infection, and acute Haemobartonella muris and Eperythro-
zoon coccides infections of rats and mice, respectively (3,4,11,12,14,15).
To our knowledge, the present study is the only one in which a causal

relationship of the agglutinin to anemia has been indicated.

REFERENCES

1. Barrett, J. T., R. M. Rigney, and R. R. Breitenbach. 1970. Char—
acteristics of the hemagglutinins produced during Plasmodium
Zophurae malaria in chickens. Infec. Immun. 2:304-308.

2. Chan, P. E. Y., and H. F. Deutsch. 1960. Immunochemical studies
of human serum Rh agglutinins. J. Immunol. 85:37-45.

3. Cox, H. W., W. F. Schroeder, and M. Ristic. 1966. Hemagglutina-
tion and erythrophagocytosis associated with the anemia of

Pflasmodium berghei infection of rats. J. Protozool. 13:327-332.

10.

11.

12.

151

Cox, H. W., and G. M. Iturri. 1973. Idiopathic immunologic
activity associated with anemia from infection with HemobartoneZZa
muris of rats and Eperythrozoon coccidOies of mice. (In press)
Infec. Immun.

Dacie, J. V. 1965.‘ Hemolytic anemia. Ann. N.Y. Acad. Sci. 124:
415-421.

Lykins, J. D., A. R. Smith, E. W. Voss, and M. Ristic. 1971.
Physical separation of three soluble malarial antigens from the
serum of chickens infected with Plasmodium gaZZinaceum. Am. J.
Trop. Med. Hyg. 20:394-401.

Ludford, C. G., R. M. Corwin, H. W. Cox, and T. A. Sheldon. 1969.
Resistance of ducks to a Plasmodium sp. induced by a filterable
agent. Milit. Med. (Suppl.) 134:1276-1283.

Ludford, C. G., H. G. Purchase, and H. W. Cox. 1972. Duck infec-
tious anemia virus associated with Plasmodfium Zophurae. Exp.
Parasitol. 31:29-38.

McGhee, R. B. 1960. An autoimmune reaction produced in ducklings
in response to infection of duck embryo blood infected with
Plasmodium Zophurae. J. Infect. Dis. 107:410-418.

McGhee, R. B. 1964. Autoimmunity in malaria. Am. J. Trap. Med.
Hyg. 13:219-224.

Musoke, A. J. 1973. Immunologic and pathologic studies of infec-
tions with rat adopted Plasmodium chabaudi. M.S. Thesis, Michigan
State University.

Oki, Y., and K. Muira. 1970. Characteristics of red cell auto-
antibodies in equine infectious anemia. Jap. J. Vet. Res. 32:

217-227.

13.

14.

15.

16.

17.

18.

19.

152

Ristic, M. 1961. Studies on anaplasmosis. III. An autoantibody
and symptomatic macrocytic anemia. Am. J. Vet. Res. 22:871-876.

Schroeder, W. F., and M. Ristic. 1965. Anaplasmosis. XVII. The
relation of autoimmune process to anemia. Am. J. Vet. Res. 26:
239-245.

Schroeder, W. F., H. W. Cox, and M. Ristic. 1966. Anemia, para-
sitemia, erythrophagocytosis and hemagglutinins in Babesia
rodhaini infection. Ann. Trop. Med. Parasitol. 60:31-38.

Soni, J. L., and H. W. Cox.' l973a. Pathogenesis of acute avian
malaria. I. Immunologic reactions associated with anemia,
splenomegaly, and nephritis of acute Plasmodium gaZZincaceum
infections of chickens. In preparation.

Soni, J. L., and H. W. Cox.' l973b. ~Pathogenesis of acute avian
malaria. 11. Study of antigens and antibodies associated with
anemia of acute Plasmodium gaZZinaceum infection of chickens.

In preparation.

Thoongsuwan, S., and H. W. Cox. 1973. Antigenic variants of the
hemosporidian parasite, Babesia rodhaini, selected by
treatment with immune globulin. (In press) Ann. Trop. Med.
Parasitol.

Zuckerman, A. 1960. Autoantibody in rats with Plasmodium berghei.

Nature 185:189-190.

153
Table 1. Average titers of cold-active agglutinin for trypsinized human
type "0" erythrocytes in the plasma of 5 chickens with acute Plasmodium
gallinaceum infection, 3 normal chickens and 4 hyperimmunized chickens,
before and after absorption with the trypsinized cells at 4 C, the
titers of the agglutinin in saline used to elute cells at 37 C, and
tests of each material for agglutinin activity after 2-mercaptoethanol

treatment (2-ME).

 

Titers Before and After 2-ME

 

 

 

 

‘ Infected +__ Normal Hyperimmune
Material Tested Before After Before After Before After
Whole plasma 122 0 8 0 64 0
Absorbed plasma 0 0 0 0 0 0
lst 37 C eluate from
absorbing cells 51 0 4 0 16 0
2nd 37 C eluate from
absorbing cells 11 0 0 0 0 0

3rd 37 C eluate from
absorbing cells 0 0 0 0 0 0

 

154
Table 2. Average red blood cell counts (RBC x 106) on 4 chickens
(Exptl.) injected with cold agglutinin absorbed from plasma of chickens
with acute Plasmodium gaZZinaceum infection with trypsinized human type
"0" erythrocytes, and on 4 chickens injected with control materials from

normal chicken plasma (Control).

 

 

 

Days Post Av. TBC Count x 106/cmm

Injection Exptl. Control % RBC Loss t value P
O 3.23 i 0.50 3.21 :_0.42 -0.6' 0.06 N.S.
l 2.51 :_0.54 2.94 i 0.31 14.6 1.45 N.S.
2 2.46 :_0.46 3.27 :_0.30 24.8 2.90 <0.025
3 2.49 i 0.26 3.27 :_0.18 23.8 4.79 <0.005
4 2.54 i 0.57 3°25.i.0'23 21.8 2.28 N.S.
5 2.67 i 0.73 3.13 :_0.14 14.6 1.22 N.S.
6 2.70:0.75 3.14:0.14 14.0 1.14 N.S.
7 2.83 :_0.60 3.22 :_0.16 12.1 1.26 N.S
8 3.07 i 0.32 3.24 :_0.19 5.2 1.27 N.S.
9 3.23 i 0.36 3.28 i 0.21 1.0 0.24 N.S.

 

155
Table 3. Average red blood cell counts (RBC x 106) on 4 chickens injected
with plasma of chickens with acute Plasmodium gaZZinaceum from which cold-
active agglutinin had been absorbed with trypsinized human type "0"
erythrocytes (Exptl.), and on 4 chickens injected with normal plasma

treated with the trypsinized cells (Control).

 

 

Days Post Av. RBC Count x 106/cmm
Injection Control Exptl. % RBC Loss t value P
0 3.15 i 0.14 3.19 i 0.20 -l.0 0.02 N.S.
1 3.24 i 0.07 2.77 :_0.03 14.5 10.90 <0.001
2 3.21 :_0.07 2.67 i 0.22 16.8 4.37 ' <0.005
3 3.20 i 0.11 2.24 i 0.60 30.0 3.14 <0.025
4 3.26 i 0.12 2.35 i;0.34 27.7 5.01 <0.005
5 3.18 i 0.01 2.42 :_0.21 23.9 6.95 <0.001
6 3.22 i 0.09 2.57 :_0 13 20.2 9.37 <0.001
7 3.19 i 0.03 2.70 1:0 08 15.3 10.86 <0.001
8 3.20 i;0.04 2.85 1:0 09 10.9 6.86 <0.001
9 3 21': 0.05 2.92 1:0.16 9 3 3.36 <0.025
10 3.26 i 0.04 3.07 i_0.15 5 8 2.28 N.S.

 

156
Table 4. The titers of cold-active agglutinin (CA), serum antigen (SA)
and antibody to serum antigen (ABSA) in plasma from chickens with acute
Plasmodium gaZZinaceum infection, before and after absorption with

trypsinized human type "0" erythrocytes.

 

 

Chicken Cont. Cont. Cont.

Numbers 1451 1446 820 1351 831 l. 2. 3.
CA 128 64 32 256 128 8 8 8

Pre-

Absorption SA 512 128 256 128 512 0 0 0
ABBA 512 64 128' 64 128 0 0 0
CA 0 0 O 0 0 0 0 0

After

Absorption SA 512 128 256 128 256 0 0 0
ABSA 512 64 128 64 128 0 0 0

Eluates CA 32 32 32 128 32 4 4 4

from

Trypsinized SA 0 0 0 0 0 0 0 0

Erythrocytes

ABSA 0 0 0 0 0 0 0 0

157
Figure 1. 'The titers of cold-active agglutinin for trypsinized
human type "0" erythrocytes in column fraction samples after column
chromatography with Sephadex G-200 of globulin from chickens with acute

Plasmodium gaZZinaceum infection.

 

158

H muawfim

«02 most mzogu 351.00

in!

83.1”. VH
g on

$3

8

 

zoF<Ezuozoo 2.5.05.

0 0.! On. ON. 0:

cut» <1 ......

 

ON

a
.
a

—

r

 

r-..__-_.-.J

 

0. «2 o. s.
-' O O O
um 082 1v uISNz-Ia ‘IVOlldO

N

”I

 

“T

 

159
Figure 2. The serologic and physicochemical properties of cold-
active agglutinin absorbed from the plasma of chickens with acute Plas-
modium gaZZinaceum malaria at 4 C with trypsinized human type "0"
erythrocytes and eluted from the cells at 37 C.

2.1. Reactions of cold-active agglutinin (CA) with anti-chicken
globulin (ACG) showing 2 precipitin lines and a single line with anti-
chicken 7S globulin (A78). No reaction was observed with absorbed

eluate from normal chicken plasma (NC).

2.2. Immunoelectrophoresis with anti-chicken globulin of Sephadex
G-200 column fractions 50-61 (1) and 62-70 (2) from globulin of
malarious chickens, both of which contained cold agglutinin activity.
Pool 1, lower well, migrated as nearly pure 19S globulin. Pool 2,
lower well, exhibited migration patterns consistent with 19S and 7S
globulins.

2.3. Immunoelectrophoresis with anti-chicken globulin of cold
agglutinin eluted from trypsinized erythrocytes after absorption of
malarious plasma at-4 C and elution at 37 C. Eluate from malarious
plasma (1), top well, indicated the presence of protein migrating as
198 and 7S globulin. Eluate from normal plasma (N), top well, also gave
evidence of containing serum globulin. The eluate from malarious plasma
after cleavage with 2 mercaptoethanol (ME), lower well, did not give
evidence of globulins.

2.4. Disc electrophoresis stained for protein of eluate absorbed
from malarious plasma (1) and eluate absorbed from normal chicken plasma
(N). The arrow indicates a band in the range of 198 globulin that was

not seen in the control material.

2.5. Immunofluorescent study of erythrocytes from chickens after
injection of cold agglutinin. Reactions of conjugated anti-chicken
globulin with cells from an agglutinin injected bird (I) and a bird
injected with eluate absorbed from normal chicken plasma (C) are shown.
Cells did not react with conjugated antibody to any of the antigens

found in malarious blood.

160

 

5‘

 

 

Figure 2