The incidence of toxoplasmosis in a mentally retarded population
by Marlene Joyce Mackie
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Microbiology
Montana State University
© Copyright by Marlene Joyce Mackie (1970)
Abstract:
Serums from 478 mental defectives from the Boulder River School and Hospital in Boulder, Montana
were tested for antibodies for Toxoplasma gondii using the Indirect Fluorescent Antibody Test (IFAT)
and the Indirect Hemagglutination Test (IHA). Serums for a control population consisting of specimens
for Billings and Helena, Montana were tested concurrently. All populations were separated according
to age and sex. In the control populations, the IFAT showed an increase in the percent of serums
positive from ages 20-40 years. The age groups from 30-50 years showed a high percent positive in the
IHA test. The Boulder group showed no rise in incidence until age 50 years in the IFAT while the IHA
test showed a more even distribution in all age groups. In all three groups, the percent of serums
positive for each sex was essentially no different. Agreement between the IFAT and the IHA1test was
38 percent in Helena, 43 percent in Billings, and 36 percent in Boulder. In the mentally defective
group, the patients positive by the IFAT or IHA test were separated according to time in residence at
the institution and type of mental retardation. The results showed little variation of the percent of
positive serums in the length of time in residence or in the type mental retardation. Elevation and
yearly rainfall statistics for Boulder, Helena and Billings, Montana were compared with the percent
positive serums in these areas. The results showed essentially no difference. Statement of Permission to Copy
In presenting this thesis in partial fulfillment of the require­
ments for an advanced degree at Montana State University, I agree
that the Library shall make it freely available for inspection.
I
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for scholarly purposes-may be granted by my major professor, or, in
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It is understood that any
copying or publication of this thesis for financial gain shall not be
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THE INCIDENCE OF TOXOPLASMOSIS IN A MENTALLY RETARDED POPULATION
by
MARLENE JOYCE MACKIE
A thesis submitted to the Graduate Faculty in partial
fulfillment of the requirements for the degree
of
MASTER OF SCIENCE
in
Microbiology
Approved:
Chairman, Examining(Committee
Grad­
uate Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
August, 1970
iii
ACKNOWLEDGEMENT
The author wishes to express her gratitude and appreciation to
Dr. Alvin G. Fiscus for his time and guidance while working on this
project.
She would like to thank Dr. J. Allen Miller and the labor­
atory staff of St. Peter's Hospital and Montana Crippled Children's
Hospital in Helena, Montana and Bynum Jacksop and his staff at St.
Vincent's Hospital in Billings, Montana for their assistance in
collecting of the serums,comprising the control populations.
Also,
this project could not have been completed without the cooperation
and advice given by Dr. Phillip Pallister and the assistance of his
staff at the Boulder River School and Hospital, Boulder, Montana.
She would also like to thank Dr. Alvin G. Fiscus, Dr. D. G.
Stuart, Dr. W. D. Hill and Dr. S . Rogers for their advice in prepar­
ing this manuscript.
This research was supported by Public Health Service Grant,
AHT-68-034 and Grant 379, Department of Botany and Microbiology,
Montana State University, Bozeman, Montana.
iv
TABLE' OF CONTENTS
Page
VITA
........................................................... ii
ACKNOWLEDGEMENT . . . . . . . . . . . . . . . . . . . . . . . . .
iii
TABLE OF CONTENTS ............................................... iv
LIST OF TABLES
.................
vi
LIST OF F I G U R E S ..................
ABSTRACT
vii
..................................................... viii
I N T R O D U C T I O N ..............
MATERIALS
I
AND M E T H O D S .....................
Study groups
Group I
. ......................
15
...................... ; ................. 15
Group 2
Group 3
............... 15
...................... .. .
.................. . . ; ................ .
Maintenance of the organism . . . . .
Indirect Fluorescent Antibody Test
Antigen preparation
........
..................
..............
Preparation of the anti-human
Antigen preparation
16
16
.
. . . . . . . .
conjugate. . . . . .
Indirect Fluorescent AntibodyTest procedure
Indirect Hemagglutination Test
. . . . .
16
....
17
17
18
21
.........................
23
..............................
23
Tanning of the sheep red blood c e l l s .......... 24
v
'Page
RESULTS
Sensitization of the tanned sheep red
blood cells ......................................
24
Technique of the microtitration test . . . . . . . .
25
............................................ ..
26
The incidence of toxoplasmosis correlated
to age and sex .....................................
26
Correlation between length of time institution­
alized and incidence of toxoplasmosis ............
31
Incidence of toxoplasmosis correlated to the
typq of mental retardation .........................
31
Correlation of toxoplasmosis with elevation
and rainfall .......................................
34
DISCUSSION
36
SUMMARY ,
BIBLIOGRAPHY
43
vi
LIST OF TABLES
Page
Table
Table
Table
Table
Table
Table
Table
I
II
III
IV
V
VI
VII
Incidence of Toxoplasma antibodies in
various geographical locations ..........
...
12
The total number of serums tested in
the 3 test groups , . ........................
27
The total number of serums tested in
each group ................. . . . . . . . . . .
28
Percent agreement between the Indirect
Hemagglutination Test and the Indirect
Fluorescent Antibody T e s t .............. ..
29
Correlation between incidence of positive
serums and the years in residence at.
Boulder River Training School and Hospital . . .
32
Indirect Fluorescent Antibody Test and
Indirect Hemagglutination Test positive
patients separated according to diagnosis
of type of mental r e t a r d a t i o n .......... ..
33
Correlation of incidence of toxoplasmosis
with elevation and rainfall in the three
geographical locations ..................
35
...
vii
LIST OF FIGURES
,
Page
Figure I
Pattern of congenital toxoplasmosis. , ........
6
Figure 2
Toxoplasma gondii as it appears in the
Indirect Fluorescent Antibody Test . , ........
22
Figure 3
The Indirect Hemagglutination Test
for toxoplasmosis............ . . r ........ 22
Figure 4
Distribution of patients testing positive
according to the Indirect Fluorescent Anti­
body Test (IFAT) and the Indirect
Hemagglutination T e s t .......... .. ...........
30
viii
ABSTRACT
Serums from 478 mental defectives from the Boulder River School
and Hospital in Boulder, Montana were tested for antibodies for '
Toxoplasma gondii using the Indirect Fluorescent Antibody Test (IFAT)
and the Indirect Hemagglutination Test (IHA). Serums for a control
population consisting of specimens for Billings and Helena, Montana
were tested concurrently. All populations were separated according
to age and sex. In the control populations, the IFAT showed an in­
crease in the percent of serums positive from ages 20-40 years. The
age groups from 30-50 years showed a high percent positive in the IHA
test. The Boulder group showed no rise in incidence until age 50 years
in the IFAT while the IHA test showed a more even distribution in all
age groups. In all three groups, the percent of serums positive for
each sex was essentially no different. Agreement between the IFAT
and the IHAltest was 38 percent in Helena, 43 percent in Billings,
and 36 percent in Bowlder. In the mentally defective group, the
patients positive by the IFAT or IHA test were separated according
to time in residence at the institution and type of mental retard­
ation. The results showed little variation of the percent of positive
serums ip the length of time in residence or in the type mental re­
tardation. • Elevation and yearly rainfall statistics for Boulder,
Helena and Billings, Montana were compared with the percent positive
serums in these areas. The results showed essentially no difference.
INTRODUCTION
In 1908, Toxoplasma gondii was isolated by two different groups
working independently.
Nicolle and Manceaux (1908) isolated the
organism from Ctenodactylus gondii, a rodent in Africa and Splendore
(1908) isolated it from rabbits in Brazil.
In 1908, Darling isolated
cyst forms from the arm muscle of a Barbadian negro.
From 1908 to
1939, data were published which dealt primarily with morphological
descriptions and the occasional occurrences in various animals and
man,(Feldman, 1968a).
Wolf et al. (1939) demonstrated that the
organism invaded the fetus in utero resulting in a stillborn or
diseased infant.
Two years later, acquired toxoplasmosis was describ­
ed in children as an encephalitis by Sabin and in adults as a spotted
fever syndrome by Pinkerton and Henderson (1941).
Since that time,
numerous reports citing toxoplasmosis as a multisystem disease
(Altman, 1968) have emanated from North and South America;; ,Europe,
Africa, Asia, and Oceania (Wright, 1957).
Toxoplasma is an obligate, intracellular, parasite (Sabin and
Olitsky, 1937) able to ipfect a wide range of animal hosts including
marsupials, moles, rodents, carnivores (Moller, 1952), swine (Farrell
et al., 1952), sheep, cattle (Jacobs, 1960b), various simians (Sabin
and Ruchman, 1942; Cowen and Wolf, 1945), birds (Jacobs and Jones,
1950; Jacobs et al., 1952) and some reptiles (Frenkel, 1953; Jacobs,
1956).
2
It proliferates in all cells in the vertebrate host with the
exception of the non-nucleated red blood cells (Feldman, 1968)-.
Entry into the cell is facilitated by the use of lysozome-like
structures and hyaluronidase (Norby et al., 1968; Lycke et al., 1965).
Once inside the host cell, Toxoplasma is capable of competing with
thq host cell for nutrients and metabolites (Jacobs, 196,3).
Eventu­
ally, this process results in host cell rupture with the resultant
spread of virulent organisms to other cells (Jacobs, 1963).
A
variation of the virulent life cycle often occurs when cysts develop
in a vacuole of the host cell (Jacobs, 1967).
As this occurs, the
proliferation of the organism subsides, the symptoms of the disease
disappear, leaving the host with chronic toxoplasmosis for life.
The
condition does not incapacitate the host but symptoms may recur when
a cyst ruptures releasing viable organisms which are able to reinfect
susceptible cells (Jacobs, 1963).
More recent evidence indicates
that the chronic state may not be induced by cyst rupture only, but
by the persistence of residual trophozofte.gr-. in trace! Iular Iy without
host cell rupture (Jacobs, 1967).
In summary, the characteristics of Toxoplasma which are crucial
in its' pathogenicity are: (l) its' ability to grow in many types of
cells; (2) its' ability to form a cyst; and (3) its' obligate, intra­
cellular development (Altman, 1968).
3
Early studies on transmission of Toxoplasma gondii centered
about the trophozoite;-
The organism was found to be fragile, not
resistant fo osmotic pressure changes, and incapable of surviving
for long periods extracellularIy (Jacobs, 1968).
Since the acute
i
disease is characterized by a parasitemia, investigations were
directed toward blood sucking arthropods as reservoirs of the disease.
Mosquitoes, fleas, triatomid bugs, bedbugs, lice, mites, and ticks
were tested with no success (Jacobs, 1968).
Large numbers of trophozoites introduced directly into the
stomach of the recipient host failed to produce the disease while
the feeding of tissue from chronically infected animals did (Jacobs,
1968).
This initiated studies which isolated the qyst from animal
tissue, showed its * incidence and characterized its* resistant
qualities (Jacobs et al., 1960a; Work, 1968).
Data published by
Jacobs et al. (1960a) and Work (1968) showed that the cyst is resist­
ant to gastric juices for 2-3 hours, resistant to freezing, but
susceptible to heat.
They also showed that the viable trophozoites
released from a cyst are capable of surviving in an environment
simulating the small iptestine, firmly establishing the feasibility
of.the opal route of transmission through undercooked meat (Kean
et al, , 1969).
Although this offered a partial explanation, addition­
al information was needed since many animals and human populations
4
which are strict herbivores (Jacobs et al., 1954b; Jacobs et al.,
1960b) have a high incidence of toxoplasmosis.
Research initiated
in 1968 by Hutchinson et al. linked transmission of the organism with
the egg of the nematode, Toxocara cati, in the feces of an infected
cat.
However, further studies showed that it is possible to transmit
Toxoplasma in the absence of a nematode infection (Frenkel et al.,
1969; Sheffield and Melton, 1969).
Work and Hutchinson (1969) have
since isolated what appears to be a new cyst form, from fecal floats
of infected cats, which is able to produce the disease whep inoculated
into laboratory mice intraperitoneally.
The oral route of trans.-..'
mission could explain the widespread incidence in populations with
poor sanitation standards (Remington, 1970).
In addition to the modes of transmission discussed above,
congenital transmission via the placenta has long been established in
humans and animals (Wolf et al., 1939; Remington et al., 1961; Jacobs,
1968).
The syndromes caused by Toxoplasma gondii are extremely varied
but can be placed into two major groups, congenital and acquired.
Congenital toxoplasmosis occurs as an accidental complication of
an inappafent primary infection of a pregnant human (Feldman and
Miller, 1956b).
The manifestations in the infant depend on the
virulence of the org-anism, the resistance of the mother, and the
5
stage of pregnancy (Feldman and Miller, 1956b).
If the infection
occurs in the first trimester of pregnancy, fetal death is most
common; if it occurs during the second trimester, the result is
widespread disease causing infant death or such severe conditions as
hydrocephalus, microcephalia, psychomotor retardation, cerebral
calcifications, chorioretintis or microphtalmus.
Infections occur­
ring during the last trimester produce acute symptoms of encephalitis
with fever, generalized convulsions, and various paralyses and viceral
involvement ill the form of jaundice, anemia, purpura, petichiae,
%
lymphadenopathy, hepatosplenomegaly, and chorioretinitis (Altman,
1968).
The pattern of congenital toxoplasmosis is outlined in
Figure I (Macer, 1963).
Of the infants infected congenitally, one-fourth are premature
with a mortality rate of 27 percent.
Of those babies carried to
term, 12 percent die and of those that survive, 80 percent are
defective (Altman, 1968).
Feldman (1953) found psychomotor retard­
ation in 56 percent, hydrocephalus or microcephalia is 33 percent
-r
each, convulsive disorders in the first three years in 50 percent,
chorioretinitis in 80-90 percent with bilateral involvement in 85
percent, and cerebral calcifications in 63 percent of youngsters who
tested Methylene Blue Dye test positive (Altman, 1968; Sabin and
Feldman, 1948).
It is important to note that often the only evidence
6
I
I
Mother (may be asymptomatic)
Parasitemia.
Spontaneous abortion
MongoIismj
Same infants
appear normal
at birth
I
-i
Gradual increase
in head size
Tetrad of
Congenital
Toxoplasmosis
Figure I.
hydrocephaly
convulsions
chorioretinitis
cerebral calcifications
Microcephaly
Hydrocephaly
Icterus
Rash
Hepatosplenomegaly
Splenomegaly
Lymphadenitis
Convulsions
Chorioretinitis
Pattern of congenital toxoplasmosis.
7
of congenital toxoplasmosis is the ocular involvement and that the
acute, fulminating disease producing the classic tetrad of symptoms
including chorioretinitis, hydrocephaly or microcephalia, psycho­
motor retardation and cerebral calcifications is much less common
than the subacute.-asymptomatic form (Fair, 1959; Franke, 1960).
Feldman and Miller (1956) generalized the following about con­
genital toxoplasmosis: (l) transmission can take place in any season
(2) a recent infection is usually observed in the mother but rarely
in the father; (3) sex of the fetus was not a factor; (4) twins are
usually both infected, but not to the same extent; (5) mothers who
have antibodies before conception do -not produce congenitally in­
fected children; (7) not all intrapregnancy infections lead to fetal
disease and some fetuses escape infection altogether; and (8) subse­
quent children are not congenitally infected.
The last generaliz­
ation has been disputed by researchers who feel that his may not
always be the case.
They stress that chronic toxoplasmosis is an
important cause of repeated spontaneous abortions, stillbirths, and
premature deliveries (Feldman, 1968b).
Acquired toxoplasmosis presents an even more diverse symptomologic picture.
The four manifestations of the disease are: (l) ,
lymphodonosa , (2) exanthematica, (3) cerebrospinalis, and (4) Opthalmica (Siim, 1956).
The most common is the lymphatic fonp.
The
8
onset may be acute or subacute and the course may be febrile,
afebrile, or subclinical (Feldman, 1968b).
Non-specific symptoms
such as headaches, nausea, abdominal pain, arthralgias and myalgias
may be present during the course of the disease (Feldman, 1968b),.
All nodes, including hilar, mesenteric, and retroperitoneal may be
involved.
Biopsy of the node reveals typical forms of Toxoplasma
which when inoculated into laboratory animals will produce the <_
disease (Siim, 1956; Feldman, 1968b).
This form of the disease may
mimic many other diseases such as infectious mononucleosis (Remington
et al. , 1962), glandular fever (Siim, 1956), Hodgkin’s disease
(Jacobs, 1967) and lymphadenopathy of unknown origin (Siim, 1956)
and for this reason is often misdiagnosed.
Immunity against the organism consists of macroglobulin (19S)
which occurs in both the acute congenital or acquired syndromes.
7S antibodies are demonstrated only in infants born to chronically
infected mothers (Remington et al., 1966).
IBe macroglobulins
pro­
vide the basis for many of the serologic tests presently used in
diagnosis of the disease.
The Methylene Blue Dye test developed by Sabin and Feldman in
1948 was the first serologic tool for the diagnosis of toxoplasmosis.
However, the inconvenience of dealing with live parasites and the
difficulty in obtaining large amounts of the "accessory" factor,
9
found only in fresh human serum, initiated a search for new methods
of testing.
A complement fixation procedure was developed in 1954 by Awad
(Eichenwald, 1956).
The test was not a useful diagnostic tool be­
cause antibody titers did not rise until late in the course of the
disease and receded after a short time, making it difficult to obtain
an early diagnosis.
\ A hemagglutination test was developed in 1957a by Jacobs and
Lunde.
The procedure showed promise'-because of the stability of the
antigen and the rapid reading of the test results (Jacobs and Lunde,
1957a).
Additional adaptation to microtechniques has further
simplified it by increasing the rapidity of performance and decreas­
ing the reagent demand (Lewis and Kessel, 1961).
Other tests have been described including a direct agglutination
test (Fulton and Turk, 1959) and a flocculation test using acrylic
particles (Siim and Lind, I960), however, these have not been used
frequently.
In 1959, Carver and Goldman reported a method of staining
Toxoplasma gondii present in tissue sections with fluorescein labeled
antibody.
This work led directly to the development of the Indirect
Fluorescent Antibody test (Kelen et al., 1962).
The procedure was
further developed and the specificity validated by Fletcher in 1965,
10
Sulzer and Hall in 1967, and Chessum in 1970.
They found that the
test was as sensitive as the Methylene Blue Dye test but more
desirable because it had a sharpened point, stable reagents which
were easily prepared and standardized,, and there was no need.for live
parasites or the "accessory",factor.
A skin test using a water-lysed suspension of organisms relies
on the production of a delayed type of hypersensitivity.(Jacobs,
1963).
It is used primarily in epidemiological studies where large ,
numbers of people are tested.
Finally,, toxoplasmosis can be diagnosed through isolation of
the organisms from the tissues (Walls et al., 1963) and inoculation
into laboratory animals with the resultapt production of the disease
(Eichenwald, 1956).
Although there is a generalized parasitemia
present during the acute disease, isolation procedures frequently
fail (Remington et al., 1962).
The methods discussed are used in the diagnosis of acquired
toxoplasmosis.
They may be used in the congenital form but more
often conclusive diagnosis is made by the demonstration of the para­
site in Wright stained smears of the newborn’s spinal or ventricular
fluid.
Serological studies at the time of birth and 3 months later
should be performed to confirm the diagnosis (Feldman, 1968b).
11
Newer methods in serological diagnosis and the development of
the i'ntradermal skin test have facilitated studies on the incidence
of Toxoplasma in all parts of the world.
Table I lists some of these
studies.
Further research has been done in an attempt to correlate the
incidence of Toxoplasma antibodies with geographical location (Walls
and Kagan, 1967b; Walls et al., 1967a; Paul et al., 1964), occupation­
al associations (McCulloch et al., 1963; Walls and Kagan, 1967b;
Kabayashi, 1963; Murakami, 1964), urban versus rural residence
(French et al., 1970; Gibson, 1956; Schnurrenberger et al., 1964),
animal contact (Schnurrenberger et al., 1964; Kijriball et al., 1960),
and climatological variations such as altitude and rainfall (Walton
et al. , 1966b).
Generalizations drawn from these studies are; (I)
the incidence of antibodies tends to increase with age; (2) a higher
incidence occurs in geographical locations with a lower elevation
and a greater rainfall; (3) a higher incidence occurs in rural
populations; (4) persons in contact with animals or animal products
have antibodies more frequently than those who are not; (5) con­
sumption of raw beef or eggs increases the incidence of antibodies;
(6) the incidence was not influenced by sex.
Several groups (Burkinshaw et al., 1953; Cook and Derrick, 1961;
Fair, 1959; Kongo et al.., 1964) have published data on the occurrence
Table" I.
Author
Feldman and
Miller
M
!I
Il
I!
M
Il
Il
Il
II
Incidence of Toxoplasma antibodies in various geographical locations.
Date
Country
Locality
1956
1956
1956
USA
USA
Iceland
Alaska
Southwest
USA
USA
USA
USA
Haiti ■
Honduras
Tahiti
USA
USA
Costa Rica
USA
USA
Guatemala
Mexico
USA
USA
Portland, Ore
St Louis, Mo
New Orleans
Pittsburgh
1956
1956
1956
1956
1956
1956
1956
Gibson et al„ 1956
Jacobs et al = 1954
1958
Gibson
Gibson
1956
Gibson
1956
Gibson
1958
Unpub
Biagi*
Jacobs et aI* Unpub
1965
Feldman
Tennessee ■
New York City
Turrialba
Memphis, Tenn
Memphis, Tenn
Esciunta
Tampico
Washington,DC
Northeast
North MidAtlantic
South Atlantic
East North
Central
East South
Central
Type of
group
Eskimo
Navdjo
Normal
pop.
Il
ft
Il
Il
Il
It
Il
Test used
No.
tested
No. +
%.+
Dye test
Dye test
Dye test
21
236
108
0
10
12
0
4
11
Dye test
Dye test
Dye test
Dye test
Dye test
Dye test
Dye test
Dye test
Dye test
Dye test
Dye test
Dye test
Dye test
Intradermal
Dye test
293
184
270
144
104
266
121
987
62
156
317
700
100
231
124
51
47
84
51
37
170
82
215
9
138
75
42
94
HO
33
17
26
31
35
36
64
24
6
94
48
27
109
22
20
193
469
30
18
16
18
549
97
18
256
49
19
Negro
Jewish
Adults
Adults
Children
> 16
All ages
Negro
Military
recruits Dye test
68
22
15
89
Table I' (Continued)*
Author
Date
Country
Locality
West North
Central
West South
Central
■Mountain
Pacific
Lamb and
Feldman
1968
Brazil
Midtvedt
Ludlam
Wallace
1965
1965
1969
Norway
Africa
Pacific
Atolls
Kobayashi
et al„
Murakama
* Wright3 1957
1963
Japan
164 ■ Japan
All -parts
Niger Delt
Eauripik
Waluae
Ifalik
Tokyo
Entire country
Type of
group
Test used
' .--
Military
recruits Dye test
Children Dye test
All ages Dye test
All ages Dye test
All ages Dye test
All ages Dye test
Abattoir
workers Intradermal
Pluck
handlers Intradermal
Ham making
workers Intradermal
Normal res.
"
20=24 yrs. Dye test
No,
tested
No. +
%'.+
315
37
12
201
182
406
27
6
32
13
3
8
1455
320
64
751
36
40
52
11
65
134
99
105
9
43
59
7
43
56
90
61
68
137
46
34
-84
1-81
25
54
35
30
30
7
483
14
of Toxoplasma antibodies in the residents of schools for the mentally
retarded.
Generally they found that Toxoplasma as a cause of mental
deficiency ' was rare.
All studies, except one in Georgia, had been
done outside of the United States.
Due to the fact that only one epidemiological study in a school
for the mentally retarded had been done in the United States, it was
advisable to undertake this type of study in Montana.
Antibody titers as determined by the Indirect Fluorescent Anti­
body Test and the Indirect Hemagglutination Test were correlated to
the type of mental retardation, age, sex, and length of time insti­
tutionalized.
Control populations were tested concurrently to deter­
mine incidence in a normal population.
MATERIALS AND METHODS
STUDY GROUPS
Group I. .478 serums were drawn from residents selected from groups
classified according to diagnosis at the Boulder River School and
Hospital located at Boulder, Montana.
The groups included:
(1)
Chromosomal defects (XXYjXX/XO,XXY / t) and mongolism
(2)
deformity syndromes, unclassified (three minor defects or
more) and classified
(3)
genetic, including PKU, homocysteinuria , deafness, pseudo­
hypothyroidism, Mast syndrome
(4)
prematurity pf unknown cause and caused by attempted
abortion or placenta praevia
(5)
post infection, in utero, unknown, Rubella, Coxsackie B ,
poliomyelitis, mumps encephalitis, Western Equine encephal­
itis, measles encephalitis, and tubercular meningitis
(6)
genetic and environmental Including jaundice, kernicterus,
toxemia of pregnancy and unclassified
j
(7)
familial (no deformities) caused by consanguinity, socio­
cultural deprivation, mental retardation with mental ill­
ness and aphasia
(8)
trauma caused by a fall, an accident or during birth
(9)
unclassified group associated with microcephaly, spasticity,
epilepsy, and hydrocephalus and another group with mental
retardation with no apparent cause.
Residents were selected without regard to age or sex.
specimens were talcep. and the serums were frozen.
Blood
16
Group 2,
One portion of the control population consisted of 285
serums drawn from patients admitted' to St. Peter’s Hospital and
Montana Crippled Children's Hospital in Helena, Montana from September
1969 through November 1969.
The patients were selected randomly
without consideration for sex and age, except in the children's
hospital where all were under 10 years of age.
Group 3.
Another portion of the control population, consisting of
197 serums was drawn from patients admitted to St. V incentbs.-'Ho.ap.i.f41
in Billings, Montana from March 1970 through May 1970.
Again, the
patients were delected without regard to sex or age.
In selecting the control group from two different cities in
Montana, an attempt was made to demonstrate the effect of climatol­
ogical variations such as elevation and rainfall on the incidence of
the disease.
MAINTENANCE OF THE ORGANISM
: .^Toxoplasma gondii suspended in sheep red blood cells was received
from the National Communicable Disease Center, Atlanta, Georgia,
courtesy of Dr. Kenneth Walls.
This suspension was propagated by
inoculating 0.5 ml intraperitoneally (IP) into Swiss Manor mice
(Walls, 1969).
Thereafter, it was maintained by inoculating 0.1 ml
17
IP of peritoneal fluid diluted 1:100 in sterile saline into 6-8 week
old animals (Eyles and Coleman, 19563),. The organism was passaged
every 4 to 6 days (Fletcher, 1965), and was preserved by freezing
according to the procedure of Eyles et al. (1956b)\
This was
accomplished by collecting the peritoneal fluid from infected mice
and diluting it 1:5 with a solution of 20 percent serum in physiolo­
gical saline.
ampoules.
One ml samples were placed into sterile 2 ml glass
To each ampoule, I ml of a 10 per cent glycerol solution
was added, the contents mixed with a syringe, and the necks sealed
with heat.
The ampoules were frozen by placing them in a basket
which had been lined with a layer of absorbent cotton approximately
I inch in thickness.
-70°C.
The baskets were placed in a REVCO freezer at
The suspensions were warmed in a 37°C water bath for 5 minutes
and 0.5 - 1.0 ml was injected into Swiss Manor mice (Eyles et al.,
1956b).
INDIRECT FLUORESCENT ANTIBODY TEST
Antigen preparation.
Peritoneal fluid was collected from mice that
had been inoculated 4-5 days earlier.
The exudate was centrifuged at
200 rpm for 10 minutes to sediment the leucocytes.
The supernatant
was removed, phosphate buffered saline (PBS) pH 7.2 added, and
recentrifuged at 2000 rpm for 15 minutes to sediment the organisms.
18
The supernatant was discarded and the organisms were resuspended in
PBS pH 7.2.
The wash procedure was repeated 3 times after which the
organisms were suspended in a sufficient volume of PBS pH 7.2 to
obtain a concentration of 25-30 organisms per high, dry, field.
A
small drop of the suspension was placed in each of four 1.5 cm
circles inscribed on microscope slides with a waterproof, felt-tip
pen.
The slides were allowed to dry af room temperature and stored
at -70°C (Fletcher, 1965; Sulzer and Hall, 1966).
Preparation of the anti-human,
conjugate.
Fourteen human serums
were drawn from patients randomly selected at St. Peter's Hospital
in Helena, Montana.
The serums were pooled prior to their inoculation
into 3 randomly bred rabbits.
Three ml of serum was injected intramuscularly into each rabbit.
Three similar injections followed at 10 day intervals, at the end of
which the rabbits were bled from the marginal ear vein.
The precipi­
tin titers as determined by the tube test were number I, I:10^280;
number 2, 1:5280; and number 3, 1:5280.
Each rabbit was given an additional injection of I ml of human
serum intravenously in order to elevate the titer, after which they
were: number I, 1:20,560; number 2, 1:20,560; and number 3, 1:10^280
(Campbell et al., 1964).
serums stored at -15°C.
The rabbits were exsanguinated and .the
19
An aliquot of rabbit anti-human serum was diluted with 10
volumes of distilled water to which an equal volume of saturated
ammonium sulfate (Mallinkrodt) was added.
The mixture was placed
at 4°C overnight after which the ammonium sulfate was removed by
aspiration, leaving the globulins suspended in a small volume of
50 percent saturated ammonium sulfate.
A second precipitation was
done by dissolving the globulins in a volume of distilled water equal
to the volume of the precipitated globulins.
Again, an equal volume
of saturated ammonium sulfate was added to the solution.
The pre­
cipitated globulins were collected by centrifugation.
For dialysis, the globulins were dissolved in a volume of water
equal to the original volume of the whole serum.
They were refrig­
erated at 4°C for 10 minutes to dissolve all the globulins.
The
solution was placed into dialysis, tubing and dialized against 0.85
percent sodium chloride at 4°C.
Two dialyses, of 12 liters each were
performed, one from 1:00 p.m. to 5:00 p.m. in the afternoon and the
other from 6:00 p.m. to 7:00 a.m. the next morning.
At the end of
the second dialysis, the 0.857. NaCl used for dialysis was checked
with a I percent solution of barium chloride.
The absence of
turbidity indicated the elimination of the sulfate ion (Goldwasser .
and Shepherd, 1958).
The globulin solution was pipetted from the
tubing and the volume measured.
20
Carbonate-bicarbonate buffer (0.5 M pH 9.0) was added to the
globulin solution in an amount equal to 10 percent by volume.
The
fluoroisothiocyanate (FITC) added to the globulin mixture at this
point was calculated using the globulinrvolume after dialysis, the
amount of globulin present in the dialized solution as calculated
by the Biuret procedure (Bauer et al., 1968), and 20 milligrams of
FITC needed per gram of protein (Fletcher, 1965; Walls, 1969).
Conjugation was performed at room temperature for 2 hours by
shaking the dye-globulin mixture gently on a mechanical rotator.
Unconjugated FlTC was removed by filtering the conjugate through a
G-50, fine, globular Sephadex column which had been calibrated with
0.02 M PBS pH 7.2.
The conjugated serum was stored into I ml aliquots
at -15°C (Fletcher, 1965).
The optimum dilution of the conjugate was determined by making
dilutions of 1:20, 1:40, 1:80, and 1:100 in PBS pH 7.2.
The dilutions
were used in the performance of the Indirect Fluorescent Antibody Test
on known positive and negative serums.
The optimum dilution of
conjugate was that dilution which gave maximum fluorescence at the
known titer of the positive serum and negative fluorescence with the
negative serum.
21
Indirect Fluorescent Antibody Test procedure.
were diluted 1:16 and 1:64.
Initially, serums
Antigen slides were washed in a light
stream of distilled water, blotted with laboratory tissue, and placed
on wet filter paper in 15 cm petri plates.
Each smear was covered
with a different serum dilution, the pan was covered and placed in a'
37°C incubator for 30 minutes, after which the serums were rinsed with
a light stream of distilled water.
The slides were immersed in a
staining dish containing PBS pH 7.2 and rotated at 15 rpm for 15
minutes.
plates.
The slides were blotted dry, and ,replaced in the petri
At this time, the smears were covered with the optimum
dilution of anti-human conjugate which had been diluted in PBS pH 7.2.
The slides were handled as above.
After rotation, the slides were
removed, washed with distilled water, blotted dry; and coverslipped
with buffered glycerol pH 8.0 (Fletcher, 1965).
The slides were
examined on a fluorescent microscope (Leitz) using a BG-12 exciter
and an GG-I barrier filter (Helen et al., 1962).
With every test the
following controls were used: saline, a negative serum, and a positive
serum of known titer.
The positive serum was tested at four dilutions,
one below, one at, and two above the known titer.
was tested at l:64only.
The negative serum
A reaction was considered positive when ,
yellow green fluorescence was observed to extend around the entire
porifery of the organism (Sulzer and Hall, 1966), as shown in Figure 2.
22
Figure 2
Toxoplasma gondii as it appears in the Indirect
Fluorescent Antibody Test.
Figure 3
The Indirect Hemagglutination Test for toxoplasmosis.
Column A represents the positive control, column H the
negative control and columns B through G are negative
serums.
23
The titer was the highest dilution at which more than half of the
organisms exhibited this degree of fluorescence.
Interpretation of
the results was as follows: 1:16* to 1:64 indicated past exposure or
early stages of the disease followed by rising titers; 1:256, recent
exposure or a present infection; 1:1024, acute toxoplasmosis.-
If a
test serum had a titer of 1:64 during initial testing, the serum was
diluted 1:16, 1:64, 1:256, and 1:1024 and repeated (Walls, 1969).
INDIRECT HEMAGGLUTINATION TEST
Antigen preparation.
The peritoneal exudate from an infected mouse
was centrifuged in tared centrifuge.tubes for 10 minutes at 1300 rpm.
The supernatant was discarded and the sediment containing Toxoplasma
gondii was washed twice in 10 ml of cold saline. ( The Toxoplasma
suspension was centrifuged after the last saline wash and the super­
natant discarded.
The organisms were resuspended in 10 volumes (W/V)
of cold distilled water and kept at 4°C overnight.
The solution was
frozen and thawed for 5 to 10 cycles (-70°C to 37°C).
An equal volume
-of double strength saline (1.7%) was added to restore isotonicity.
The solution was spun for one hour at 30,000 x G in an. angle head
centrifuge (Beckman Model L) to remove cellular debris.
The super­
natant comprised the antigen, the dilution of which was 1:20.
The
antigen was stored at -20°C (Lunde and Jacobs, 1959; Chordi et al.,
24
1960),
Tanning of the sheep red blood cells.
Sheep red blood cells were
obtained under aseptic conditions in Alseverfs solution.
They were
washed 5 times with PBS pH 7.2, and adjusted to a I percent suspension
by adding PBS pH 7.2 to the packed cells.
An equal volume of I:100,000
dilution of tannic acid (Mallinkrodt) was added.
The solution was
mixed thoroughly and placed in an ice water bath for 15 minutes.
The
tannic acid treated cells were removed from the ice water bath,
centrifuged for 5 minutes at 1500 rpm.
The supernatant was discarded
and the cells were washed once with PBS pH 7.2 (Chordi et al., 1964).
The cells were resuspended in 4 I percent suspension in 0.6 percent
normal rabbit serum in PBS pH 7.2 (Lewis and Kessel, 1961).
Sensitization of the tanned sheep red blood cells.
The optimum
dilution of the antigen was determined by preparing four dilutions of
the antigen in PBS pH 6.4.
They were! I;50, 1:100, 1:200, and 1:400,
The cells wete sensitized with these dilutions as described below.
A tannit? acid cell control was prepared by sensitizing tanned cells
with PBS pH 6.4 only.
The optimum dilution was that dilution of
the antigen which gave the correct titer of, the known positive serum
and a negative reaction with a known negative serum (Walls, 1969).
25
Sensitization was accomplished by mixing equal volumes of tanned
cells and the optimum antigen concentration-
This mixture was allowed
to incubate for 15 minutes in a 37°C water bath.
After this period,
the cells wefe centrifuged, washed twice with 0.6 percent normal
rabbit serum (NRS) in PBS pH 7.2 and adjusted to a I percent suspen­
sion with 0.6 percent NRS (Chordi et al., 1964).
Technique of the microtitration test.
Using microtitration equip­
ment, serum dilutions of 0.05 ml in 0.6 percent NRS were made.
two-fold series of dilutions beginning at 1:2 was used.
A
The first
four dilutions were disregarded, the 1:32 dilution was used as a
tanned cell control, and the 1:64 dilution was used as the initial
test dilution.
were added.
To each serum dilution, 0.025 ml of sensitized cells
The plates were shaken immediately after this addition
and were left at; toom temperature for 2 hours.
The tests were read
as follows: a discrete button indicated a negative test and a disper­
sion pf the cells with no pattern indicated a positive test (Fulton
and Turk, 1959).
was as follows:
This is shown in Figure 3.
Interpretation of data
a titer of 1:64 to 1:128 indicated a past exposure
or early infection; 1:256 to 1:512, a recent infection; and 1:1024,
acute toxoplasmosis (Lewis and Kessel, 1961).
RESULTS
The incidence of toxoplasmosis correlated to age and sex.
Previous studies have indicated a trend toward increasing incidence
of toxoplasmosis with increasing age and that sex had no correlation.
Therefore, the age and sex of the patients in the present study which
were positive when tested by the Indirect Fluorescent Antibody Test
(IFAT) were compared with the total number tested in each of the
age groups as related to sex.
The results are presented in Table I.
In the general population, there was an increase from ages 20 to 40
years while institutionalized patients showed no consistent rise until
after age 50.
The same pattern was also shown by using the Indirect
Hemagglutination Test (IHA) (Table III) but was not as clear-cut.
The results displayed graphically in Figure 4 are similar to data
published by Cook and Derrick, 1961 and Fair in 1959.
Using both
tests, no significant differences were shown in the presence of anti­
bodies when patients were separated according to sex, nor did there
appear to be any significant differences between a normal population
and an institutionalized, mentally retarded one.
Correlation of test results showing those serums positive in
both tests, those positive in the IFAT only and those positive in the
IHA test only are included in Table IV.
Agreement between the two
ranged between 36 and 43 percent for the three geographical locations
27
Table II.
Age gtoup
in' years
The total number of serums tested in the 3 test groups.
Patients have been separated by sex and age. The number
positive and percent positive as tested by the Indirect
Fluorescent Antibody test has been included.
number tested number positive
males females males females
0 - 9
10 - 19
20 - 29
30 * 39
40 - 49
50 - 59
60 - 69
70 - 79
80 - 89
TOTAL
18
7
7
12
13
19
23
15
16
130
16
12
22
15
18
20
18
15
19
155
0 - 9
10 - 19
20-29
30 - 39
40 - 49
50 - 59
60 - 69
70 - 79
80 - 89
TOTAL
0
14
12
6
14
15
7
8
2
78
I
15.
25
21
20
20
10
4
3
119
0 - 9
10 - 19
20 - 29
30 - 39
40 - 49
50 - 59
TOTAL
17
85
85
36
32
9
267
11
75
78
30
12
4
211
HELENA
2
0
2
I
0
I
I
I
I
9
percent positive
males
females
percent
total
0
0
2
I
0
2
I
2
2
10
11.0
00
27.5
8.3
0
5.3
4.4
6.7
6.3
6.8
0
0
9.1
6.7
0
10.0
5.5
13.2
10.6
6.5
5.9
0
13.8
7.4
0
5.1
4.9
10.0
8.5
6.3
BILLINGS
0
0
2
I
0
4
I
3
0
4
I
0
I
I
I
0
0
0
10
8
0
7.2
0
16.6
28.5
0
14.2
12.4
0
10.2
0
13.4
16.0
14.3
0
5.0
10.0
0
0
8.5
0
10.3
10.8
14.8
11.8
2.9
11.8
8.4
0
9.2
5.8
5.9
9.5
2.7
6.2
22.0
7.5
9.1
. 13.3
2.6
10.0
0
25.0
8.2
7.0
9.4
6.2
6.2
4.6
32.0
7.8
BOULDER
I
I
10
5
2
8
I
3
2
0
2
I,
20
17
28
Table III.
Age group
in years
The total number of serums tested in each group. The
patidnts have been separated on the basis of sex and age.
Positive results as determined by the Indirect Hemagglut­
ination Test have been included.
number tested number positive
males females males females
percent positive
males
females
percent
total
18
7
7
12
.13
19
23
15
16
130
16
12
22
15
18
20
18
15
19
155
HELENA
I
0
0
I
0
I
2
I
I
7
0
0
0
I
I
0
I
0
0
3
5.6
0
0
8.3
0
5.3
8.7
6.7
6.3
5.4
0
0
0
6.8
5.6
0
5.6
0
0
2.0
2,8
0
0
7.4
3.2
2.6
7.6
3.4
2.9
3,5
9
19
29
39
49
59
69
79
89
0
14
12
6
14
15
7
8
2
78
I
15
25
21
20
20
10
4
3
119
BILLINGS
0
0
0
0
3
0
0
I
0
4
0
I
2
2
0
I
0
0
0
6
0
0
0
0
21.5
0
0
12.5
0
5.1
0
6.8
8.0
8.5
0
5.0
0
0
0
5.1
0
3.5
5.4
7.5
8.8
2.9
0
8.3
0
5.6
0 - 9
10 - 19
20 r- 29
30 - 39
40 - 49
50 - 59
60-69
TOTAL
. 17
85
85
36
.32
9
3
267
11
75
78
30
12
4
I
211
BOULDER
0
I
3
5
I
2
I
I
2
0
0
I
0
0
10
7
0
3.5
1.9
2.8
6.7
0
0
2.6
9.1
6.7
2.6
3.3
0
25.0
0
4.6
• 3.6
5.0
1.8
3.1
4.6
7.7
0
3.5
0 - 9
10 - 19
20 - 29
30 - 39
40 - 49
50 - 59
60 - 69
70 - 79
80 - 89
TOTAL
0
10
20
30
40
50
'60
70
80
-
Table IV.
Location
Percent agreement between the Indirect Hemagglutination Test and the
Indirect Fluorescent Antibody Test.
Total
positive
No., of serums
positive in both
tests
No. of serums
positive in IFAT
only
No. of serums
positive in IHA
only
No.
No.
No.
Percent
Percent
' Percent'
Helena
21
8
38.0
11
52.0
2
10.0
Billings
21
9
43.0
10
48.0
2
9.0
Boulder
39
,- .14
36.0
23
56.0
2
O
OO
%
Posi
30
Boulder
Helena
Billings
20
30
40
50
60
70
80
90
Age in years
Figure 4
Distribution of patients testing positive according to
the Indirect Fluorescent Antibody Test (IFAT) and the
Indirect Hemagglutination Test (IHA).
31
tested.
Forty-eight to 56 percent of the tests were positive in the
IFAT only.
Of these, all but two were positive in the first dilution
(1:16) with the two exceptions positive at 1:64.
Eight to 10 percent
of the tests were positive in the IHA test only.
The titers of these
ranged from 1:64 to 1:256 with most occurring in the lower dilution.
Correlation between length of time institutionalized and incidence of
toxoplasmosis.
Table V shows the number of patients tested and the
incidence of Toxoplasma antibodies correlated with the number of
years institutionalized.
The incidence of positive serums was evenly
distributed throughout all periods of residence.
A longer time of
residence did not appear to increase the number-; of patients with
Toxoplasma antibodies.
Incidence of toxoplasmosis correlated to the type of mental retardation.
The Boulder residents tested, were divided into groups according to
diagnosis as discussed on page 15.
Table VI includes the number tested
in each group, the number positive, and the percent positive.
No
group showed a predominant incidence over any other group except gen­
etic and environment.
This may be attributed to the small number of
serums tested in this category and therefore appears statistically
insignificant.
Of the 478 serums tested in the mentally retarded
population, 5 were considered as possible cases of congenital
Table V.
Correlation between incidence of positive serums and the years in
residence at Boulder River Training School and Hospital.
Years in residence
Number tested
Number positive
Percent positive
(3-5
59
6
10.2
6-10
160
16
10.0
11 - 19
160
10
6.3
20 - 29
65
3
4.7
30 - 39
25
2
8.0
40 - 49
9
I
11.2
478
38
7.9
TOTAL
Table VI,
Indirect Fluorescent Antibody Test and Indirect Hemagglutination Test
positive patients separated according to diagnosis of type of mental
retardation.
Group*
Description
H o . tested
Group I
Chromosomal
81
5
Group 2
Deformity syndrome
50
6
12.0
Group 3
Genetic
40
I
2,5
Group 4
Prematurity
10
0
0
Group 5
Post-infection
54
3
5.6
Group ~b
-Genetic.and environment
3
I
33.0
Group 7
Familial
79
5
6.3
Group 8
Trauma
30
2
6.7
Group 9
Unknown
130
15
Microcephalus
23
3
13.6
HydrocephaIus
8
3
37.5
18
2
11.2
478
38
7.9
Epilepsy
TOTAL
*
Groups, discussed in detail on page 15.
No, positive
.
Percent positive
6.2 .
.
11.6
-
34
toxoplasmosis.
A brief history for each of these follows:
(1)
Ten year old girl, mental and physical invalid with micro­
cephaly, multiple congenital defects, bilateral exophtalmus.
Mother ill during 1st trimester of pregnancy'. IFAT positive
at 1:64; IHA positive at 1:128.
(2)
Thirteen year old boy, partial invalid with hydrocephalus.
Mother ill during pregnancy. Infection at 7 months of age
accompanied by severe penicillin reaction. IFAT negative;
IHA positive at 1:64.
(3)
Twelve year old boy, history suggests kernicterus but
jaundice not noted, blind at birth. Mother had I premature
birth and several miscarriages before patient's birth. IFAT
positive at 1:64; IHA negative.
(4)
Twenty-seven year old man, microcephalic with congenital
cataracts. IFAT positive at 1:16; IHA negative.
(5)
Six year old boy, complete invalid, institutionalized
since six months of age, hydrocephalic. Mother ill during
pregnancy. The nature of the illness was unknown. IFAT
positive at 1:16; IHA, negative.
These cases are listed as possible since a diagnosis was not
established at birth and it is conceivable that infections were
acquired in later years.
Correlation of toxoplasmosis with elevation and rainfall.
An attempt
was made to correlate the incidence of Toxoplasma antibodies with
climatological conditions.
The variation of incidence relative to
altitude and rainfall did not appear significantly different for the
three geographical locations tested.
Table VII.
Comparative data is included in
Table VII.
Location
Correlation of incidence of toxoplasmosis with elevation and rainfall
in the three geographical locations tested.
Elevation*
Annual
Rainfall*
"No,
tested
28-5"
19
10
6.3
3.5
10
9.2
. 5.6
' 17
7.8
3.5
"No. positive
IFAT
IHA
Helena
3893
9.79 in.
Billings
3139
13.10 in.
197
18
Boulder
5165
11.90 in.
478
37 ,
'Eercent positive
IFAT
IHA
Co
Cn
*
Elevation and rainfall statistics (Dightman, 1960).
DISCUSSION
This study concurs with previously published data (Burkinshaw
et al., 1953; Cook and Derrick, 1961; Kongo et al., 1964) which showed
that the incidence of toxoplasmosis was no more frequent in a mentally
retarded than in a normal population.
Establishing a firm causal relationship for congenital toxo­
plasmosis in any particular patient was not possible with information
and procedures available to this study.
All residents, except hospit­
alized patients, are primarily restricted to the school grounds but on
occasion are allowed to go to the neighboring town, thus coming into
contact with dogs and cats.
Several of the residents (ages 10-30
years) have jobs in the school farm or dairy which brings them into
contact with cattle and chickens.
Also, most residents-. Vi'sit'their
homes and some are placed into homes to work, again providing an
opportunity to come into contact with farm or domestic animals which
have been proven to be carriers of the disease (Jacobs, 1957b; Siim,
1963; Jacobs, 1968).
One might expect isolation from contact with Toxoplasma in those
patients which had been institutionalized for long periods of time.
Except in strictly hospitalized patients, this did not appear to be
the case, since non-hospItalized patients were not in residence all
year and, as mentioned above, home visits, placement procedures, and
farm and domestic animals on the grounds provide the opportunity for
)
37
contact with the organism.
This information might explain why little
correlation between time in residence and antibody titers can be
established.
That the incidence was not higher than the normal
population in all groups, suggests that one method of transmission is
not direct contact as opportunities for case to case spread both by
the respiratory and alimentary routes are frequent in this type of
institution.
Similar studies published by Cook and Derrick (1961)
presented results very similar to the present study.
In all cases tested, the mental deficiency was produced by
factors operating before, at, or shortly following birth (see Table
VI, page 33).
However, since the data presented in Tables II and III
suggest that the incidence of antibodies does not increase until much
later in life, congenital toxoplasmosis is unlikely to be a frequent
cause of mental deficiency.
This conclusion is supported by earlier
studies done by Burkinshaw et al., (1953), Fair (1959), Cook and
Derrick (1961), and Labzoffsky et al. (1965).
Little correlation could be found between the type ofi-imental
retardation and the incidence of antibodies for Toxoplasma (Table VI,
page 33)•
Previous reports published by Burkinshaw et al.,(1953)
and Cook and Derrick (1961) support this conclusion.
According to the
information, the groups affected by genetic and environmental causes
and hydrocephalus appeared to have a much higher incidence of
38
antibodies than did any other group.
However, because of the small
number of serums tested in these groups, the figures do not appear to
be statistically significant.
A previous study published by Walton et al. (1966b) related
increased incidence of toxoplasmosis to a higher rainfall and lower
elevation.
The Variations in rainfall, elevation, and apt!body
occurrence were so slight ip the three geographical areas tested that
one can conclude that pp differences existed.
Complicating the
analysis, was the fact that the residepts in the Boulder Rfver School
and Hospital'had resided ip all parts of Moptana and two had come
from outside of the state.
Also, since background information was
not available concerning the patients drawn for the control populations,
it cannot be assumed that they had lived all of their lives ip either
Helena or Billings, Montana.
Agreement between the Indirect Fluorescent Antibody Test (IFAT)
and the Indirect Hemagglutination Test (IHA) was surprisingly low.
This may be attrfbptable to the fact that responses to two different
antigens yrere measured (Kefen et al., 1962)«
Due to the nature of the
test, the surface antigens of Toxoplasma were tested ip the fFAT while
intracellular antigens were measured in the IHA (Siim, 1968).
Studfes have been made comparing the Methylene Blue Dye test
(MBD) results with IFAT results.
Correlation between the two ranged
39
from poor (Lalpzoffsky et al., 1965) to excellent (Goldman et al.,
1962-j Walton et al.^ 1966a).
lifo data have been published comparing
IHA titers and IFAT titers, however, several studies have been
published comparing IfIA titers and MBD timers.
Since the MBD test
and the IFAT have been found to agree; well, an attempt has been made
to use these studies to compare the IFAT titers and the IHA titers
obtained in the present study.
Kagen et al. (1967) reported that less
than 4 percent of the serums which tested IHA negative were positive
by the MBD test and that those were oply at low levels of activity.
Ip the present study all except 2 serums which were positive in the
IFAT and negative in the IHA were positive only in the first dilution.
The 2 exceptions were positive in the second dilution.
Walton et al. (1966a) showed 96 percent agreement between the
IHA and MBD tests while Walls et al. (1967b) showed 83 percent
correlation.
A study done by Labzoffsky et al. (1965) reported only
26 percent agreement between the two tests.
Application of this
information to the present study which showed 36 to 43 percent correl­
ation between the IFAT and IHA test, reveals comparable data.
Frevibus studies have also shown MBD test positive serums which
are negative by IHA procedures, and others in which the reverse is
true (Feldman, f.968a).
Similar data in the present study showing
positive IHA titers accompanied by negative IFAT titers were found
40
but in all cases but one the titers were low.
the titer measured at 1:256.
In this exception,
This phenomenon may be explained by the
fact that the antibodies appear at different times during the infec­
tion.
It has been noted that MBD antibodies can be detected sooner
than IHA test antibodies, apd while both antibodies can be detected
for many years the IHA remains longer^(Jacobs, 1963;;Kagen et al.,
1967; Feldman, 1968a).
Again, assuming that the IFAT and MBD tests
measure similar antigaps, we may say that the IFAT antibody appears
sooner than the IHA antibody and the IHA antibody remains detectable
lpnger.
These findings may explain the differences, however, no
evidence to indicate that this is the case has been offered.
SUMMARY
Serums were drawn from 478 mental defectives residing in the
Boulder River School and Hospital in Boulder, Montana.
Control pop­
ulations consisting of 197 sefums from Billings, Montana and 285
serums from Helena, Montana were drawn.
The incidence of antibodies
for Toxoplasma gondii was measured in all serums by the Indirect
Fluorescent Antibody Test and the Indirect Hemagglutination Test.
Antibody incidence in the mentally defective group was correl­
ated to age, sex, years in residence at the institution and type of
mental retardation.
There was an increase in the number of patients
with Toxoplasma antibodies after age - 50"< years but no consistant
pattern preceeding that age.
Sex, the type of mental retardation and
the time in residence at the institution had no apparent effect on the
number of patients with Toxoplasma antibodies.
Since no cases of
congenital toxoplasmosis couli be validated in 478 serums,, it was
concluded that the disease is rarely a cause of mental deficiency.
The Indirect Fluorescent Antibody Test and Indirect Hemmaglut!nation Test were found to agree in 36 to 43 percent of the serums
which tested positive.
The serums drawn from Billings, Helena, and Boulder were compar­
ed in relation to elevation, rainfall and percent incidence of
Toxoplasma antibodies.
The variations in the three criteria between
the three geographical locatiops mere so slight that no differences
appeared to be present.
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Mackie, Marlene J
The incidence of
toxoplasmosis in a
gent ally retarded
population.
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