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SERQLQC'HCAL AND iMMUNOLOGICAi. STUDIES
ON SEVERAL ANIMAL TRECHGMONAS SPECXES
mm

1110325 for fine Dam of M. 5.
MiCHEGAN STATE UNWERSWY
Han-s latch Rathsnfimcfim

{$55

SEROLOGICAL AND IMMUNOLOGICAL
STUDIES ON SEVERAL ANIMAL

TRICHOMONAS SPECIES

 

by

Hans Jacob Rothenbacher

A Thesis

Submitted to the School of Graduate Studies of
Michigan State University of Agriculture md
Applied Science in partial fulfillment of

the reguirements for the degree of
MASTER OF SCIENCE
Department of Microbiology and Public Health

1955

ACKNOWLEDGEMENTS

The author wishes to take this opportunity to express his sincere
thanks to Dr. William D. Lindquist of the Department of MicrObiology
and Public Health under whose inspiration and guidance the present
study was made.

The writer also deeply appreciates the financial support of the
U.S. Fulbright commission and the scholarships provided by Michigan
state university for the past two years which made it possible for

him to conduct this investigation.

85 9311

I.

II-

III.

TIBLE OF CONTENTS

INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O 0

REVIEW OF THE LITERATURE

A. mstom O O O O O O O O O O O O O O O O O O O C O O O O

B.Sorolow.........................

1. Complement Fixation Reaction . . . . . . . . . . . .

2. PmCipitinReaCtionoooooooeeoeooeoe

3. Agglutination and imobilisation . . . . . . . . . .

h. Allergicskinreaction. . .‘.. .. . .. .'.. . .

5. Antigenic structure of the Trichomonas cell . . . .

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

A.
B.
C.
D.
E.
F.
G.

H.

sourcesofTrichomnassp...............

CultureMedia.....................

Pmparatimofthemtigen...............

Injection of experimental animals . . . . . . . . . . .

Harvesting and preparation of the sera . . . . . . . .

Agglutination reactions . . . . . . . . . . . . . . ..

Differentiation of the type of agglutination . . . . .

Cross agglutination

MULTS C O O C O O O O O O O O O O O O O O O O O O O O O O

A.

Normal serum agglutinations . . . . . . . . . . . . .

1. Rabbit 89m 0 O O. O O O O O O O O C O O O O O O O O

2.chickenserum....................

3. Horse serum . . . . . . . . . . . . . . . . . . . . .

14. Cattle serum

U'lf—‘WU

10
10
13
13
13
15
17
18
19
22
2h
25
25
25
25
25

TABLE OF (DNTENTS (com-n)

N. B. ANTISERUM AGGLUTINATIONS AND cmss . . . . . . . . . . . 26
Agglutinati ons
1.Rabbitantisera...................26
2. chicken antisera . . . . . . . . . . . . . . . . . . 32

0. SOME NOTES ON THE GROWTH RATES AND THE LONGEVITY 0F
LSPECIESINDIFFERBITCULTUREMEDIA.......... 36

'V.DISCUSSION.........................bl

v1.summmnconcwsmns.................. M4

VII. BIBLImRAPHY . O O O O O O O O O O O O O O O O O O O O O O O O 46

Table

I.
II.

III .

VII.

VIII.

X.
XI.
XII.
XIII.
XIV.

XV.

LIST OF TABLES

 

 

Page
Cross titration of the antiserum produced against
the soluble cell fraction of Trichomonas sp. (nasal
iBOlate)OOOOCOOOOOOOOOOO0.00.. 27
cross titration of the antiserum produced against
the insoluble cell fractions of Trichomonas sp.
(11888118016110).....-............. 28

Cross titration of the antiserum produced against
the soluble cell substances of 2. suis (fecal isolate). 29

cross titration of the antiserum produced against
the insoluble cell fractions of g. suis (fecal isolate). 29

cross titration of the antiserum produced against
the solublecall substances of I. gallinarum . . . . . . 30

Cross titration of the antiserum produced against
the insoluble cell fractions of 11:. gallinarum . . . . . 31

Titration of a mixture of antisera produced against

the soluble and the insoluble cell fractions of '1'.

species (nasal isolate); reaction with the homologous
mtigm O O O O O O O O O O O O O .0 O C O O O O O O O O 31

Cross titration of antiserum produced against the
soluble cell fractions of 1. species (nasal isolate). . 32

Cross titration of antiserum produced against the
insoluble cell fractions of _'I_‘_. species (nasal isolate). 33

Cross titration of the antiserum produced against
the soluble «11 substances of 1. suis (fecal isolate) . 33

cross titration of the antiserum produced against
the insoluble cell fractions of I. suis (fecal isolate) . 314

Cross titration of the antiserum produced against
the soluble cell substances of 11;. gallinarum . . . . . . 3h

 

Cross titration of the antiserum produced against
the insoluble cell fractions of '_1‘_. gallinarum . . . . . . 35

 

Average counts in millions per ml after 118 hours incu-
bation . . O O O C O O . C O O O C C O C . C O . O . C O 37

Average counts in millions per m1 after 72 hours incu-
bat-11011000000000.0000...0000000037

I. INTRODUCTION
Within the last thirty years some disease conditions in man and

domestic animals, caused by protozoa of the genus Trichomonas have

 

reached a very high degree of medical and economic importance.
Mam? workers have been trying to improve the diagnosis and the
treatment of these disease-conditions by making use of serological
and immological methods.
The main purpose for this work was to study the serological

behavior of three species or varieties of the genus Trichomonas,

 

acquired from the pig and from the turkey, by applying new serol-
ogical methods and procedures which have evolved in recent years.

Another motive for this study was to confirm the findings of
Sanborn (1951.), on the serological differences of the two trich-
omonad species occuring in the nose and the large intestine of the
pig.

Last, but not least, the author hopes to stimulate by this
study mrther research, aimed at better diagnostic methods that
will eventually lead to a more successful control of the diseases

caused by trichomonads .

II. REVIEW OF THE LITERNTURE
A. HISTORY

Since Donné's (1836), description of Trichomonas vaginalis from

 

the human female, several other pathogenic trichomonads have been
discovered.
Rivolta (1878), found the causative agent of a serious pigeon

disease, which is now named Trichomonas gallinae.

 

Kunstler (1888), and Mazzanti (1900), are given credit for having

discovered Tritrichomonas foetus in cattle. It was not until several

 

decades later, that these findings gained gradual recognition through
I!
the works of Pfenninger (1927), Riedmmller (1928), and Abelein (1929),

who established the association of Tritrichomonas foetus with early

 

abortion, pyometra, and sterility in the cow.

In the case of Trichomonas vaginalis, it was Hoehne (1916),

 

‘who adopted the view that this organism was a causative agent of
human vaginitis.

Due to the numerous reports of several non-pathogenic Erich:
gmgna§_species of man, cattle, and birds, the establishment of one

specific Trichomonas species as the etiological agent for a certain

 

disease condition evolved very slowly.

considerable confusion arose about Rivolta's (1878), discovery,
as to nomenclature, and primary pathogenicity of the described organism.
After about thirty years of controversy, trichomoniasis of the upper
digestive tract and viscera of various birds is now considered as a
distinct disease entity (Stabler, 195h).

Quite recently (Reyes, 1955),a serious intestinal disease of

chinchillas was found to be caused by trichomonads.

While the pathogenicity of the mentioned species is no longer

in doubt, saprophytic and non-pathogenic forms have been reported

up to the present from a great variety of animals, ranging from
the mammals through birds, reptiles, amphibia, fish, and molluscs,
to the insects (Trait; de Zoologie, 1952).

In this country it was Emmerson (1932), who first recognized

the presence and the wide distribution of I} foetus in cattle.

 

Since that time reports have come from all over the world. "Organ
and Beach (19b2), in a study of the geographical distribution of
bovine trichomoniasis, stated that 23 countries and 35 out of the
united States had recognized the disease. Mprgan (19h6), assumed
that trichomoniasis is present in any part of the world where cattle
are located.

Because of the improvement in our diagnostic methods, bovine
trichomoniasis today has reached its deserved recognition as a

disease of high economic importance.
B. SEROLOGY

Numerous workers have attempted to apply serological and
immunological methods as an aid for the diagnosis and the treat-
ment of this protozoan infection.

1. Complement-Fixation Reaction:

 

Riedmfiller (1932), ‘was the first to try the complement-
fixation test with bovine serum, using the carbolsalt extraction

of the peritoneal exudate of Irichomonas-infected guinea pigs as

 

an antigen. ‘While all the non-infected cattle yielded negative
results, only a small number of the known infected animals showed

a positive reaction.

Witte (193M), used a carbol-killed trichomonad extract as
an antigen for his complement-fixation test. All of the control
animals were negative, yet only a few of the infected animals gave
very weak positive results. He also reported non-specific reactions
from guinea pig sera. In a later publication (193kb), he claimed
that his test was specific for cattle sera.

with a method similar to Iitte (op.cit.), Endress (1939).
tested the sera of about 1400 infected and non-infected cattle. In
routine herd examinations, and with experimental animals, he
frequently encountered a positive reaction from non-infected animals,
and a negative reaction from infected animals.

2. lirecipifl Reaction:

 

Besides the complement-fixation test, Zeeti (19140) also tried
the precipitin and the agglutination reactions. He was working with
the sera of artificially infected guinea-pigs, rabbits, and dogs.
his antigen was similar to that of Riedmrzller (1932), a saline-
extract of the peritoneal exudate of infected guinea pigs. The
results obtained did not show a definite correlation between the
serological reactions and the infections.

Precipitinogens from the sera of inmnmized rabbits, and from
vaginal washings of infected rabbits were described in further
detail by Nelson(1937). His tests showed positive results only
in the cases of heavily infected animals.

Robertson (19141), and Morgan (19143), found that the fresh,

non-inactivated sera from various domestic animals will kill and

lyse I, foetus in lower dilutions. Inactivation of the sera des-
troyed the active substance.

3. Agglutination and immobilisation-reaction:

 

 

Agglutination of trichomonads by normal guinea pig sera was
first mentioned by”litte (193k). This weak normal agglutination
was significantly increased in the sera of guinea pigs inoculated
intraperitioneally with I: £23322. Yet‘Witte did not think that
this phenomenon was of great value for the diagnosis of the
disease in cattle, because of the high level of normal agglutinine
in cattle sera. Much later morgan (l9hh), published the normal
agglutination titres for I. foetus in the sera of 2b different
vertebrates, representing Pisces, Reptilia, amphibia, Aves, and
Mhmmalia.

Zeeti (l9h0), again confirmed'wittes (193h) findings about
the normal agglutinine in guinea pig sera, and also reported the
same for dogs. Furthermore, he described a positive agglutination
in the serum of rabbits, having been inoculated subcutaneously and
intraperitoneally with 2. foetus.

By intravenous injection of’g:£ggtgg_3ndress (1939), produced
positive agglutinine. After having compared the agglutinations of
infected and non-infected cattle, he concluded that the test was
of little diagonostic value due to the relatively high titer of
normal agglutinine in the cattle serum.

Nelson (1937, 1938). Endress (1939). Morisita (1939). Zeeti
(l9b0), Robertson (l9hl), Schneider (19h1), and Byrne (l9h2), showed

that rabbit serum had a very weak normal agglutination titer. m
the other hand, a strong agglutination titer in normal inactivated
equine serum was demonstrated by Endress (1939), Robertson (191d),
Schneider (19141), and Morgan (1914-14).

Byrne and Nelson (1939) , compared the titers between the sera
of rabbits injected intravenously, with either living or formalin-
killed I. m ”suspensions, and rabbits injected vaginally. They
found low titexs in the latter animals, and high titers in the former
group.

Using a formalin - killed Trichomonas suspension, Byme (19142),

 

described a macroscopic agglutination test. A similar agglutination
procedure was described by Morisita (1939) , who injected rabbits
intravenously with formalin - killed _'I_'_. foetus. The maximum titers
of the sera were attained 25 days after the last injection, and
on the 614th day the titers had returned to normal.

Positive agglutinine were also reported by Schneider (19111),
in the sera of three rabbits injected intravenously with living or
formalin - killed I. foetus.

According to lorgan (19146), all of the previous authors
failed to detect the immobilization reaction (Morgan, 19143), because
of the relatively small numbers of trichomonads, injected as antigen.
It was Robertson (191:1), who first mentioned this phenomenon in
her agglutination experiments. She injected rabbits with washed
and heat - killed cultures of 1. foe-Lug _ and performed agglutination

tests with the immune serum against living culture suspensions of

g. foetus and putrichomastix colubrorug; A considerable degree of

 

overlapping was noticed between the two reactions which indicated
a common antigenic structure for two different strains of _‘I_‘_. foetus,
used as antigen. No such group agglutinine were demonstrable in

sera produced against Trichomonas columbae. Immobilization was

 

found to occur in the low dilutions of the inmmne sera (with a high
concentration of the agglutinine) , characterized by a rounding up
of the living organisms, shrinkage of the protoplasm, and non-motile
flagella and undulating membrane. The agglutination was described
as a ball - like clumping of the trichomonads, which did not decidedly
change their normal shape. Loose rosettes were also described,
flagella and undulating membrane being motile.

Kerr and Robertson (191:1) made use of the agglutination test
for the diagnosis of bovine trichomoniasis. Positive titers could
never be found in animals with a normal history, or in virgin heifers.
They stressed the limitations of both the agglutination and the
immobilization reaction, in that, it was not a test on the actual

presence of a Trichomonas _- infection. It was also stated that

 

sera of heifers, vaccinated intravenously and intramuscularly proved
more potent than the sera of normally infected cows, having recently
aborted. The diagnosis was made on the basis of a differentiation
in the titer and in the kind of the agglutination of the infected
animals.

In great detail, Morgan (191:3), demonstrated the imob’ilizaiion
reaction in an experiment with eight virgin heifers. Five heifers

were injected intramuscularly with living 2. foetus. Sixteen in-

jections at three to four day intervals were given in increasing
amounts (from five to fifty million), constituting a total number
of 350 million living trichomonads per heifer. The first complete
immobilization reaction.appeared from 16 to 22 days after the first
injection in a serum titer of 1:1. The maximum level of 1:128 was
reached forty to 76 days after the first injection. The total
duration of the immdbilisation titer varied from five to 105 days
in five heifers. A challenging reinjection after 102 days brought
all titers up to a positive reaction again. Two heifers injected
intravenously'according to the same schedule produced slightly
higher serum reactions than the ones, injected intramuscularly.

Two other heifers, injected intramuscularly with formalin - killed
organisms showed relatively lower titers than the heifers, injected
with living trichomonads. At no time during the experiment could
living trichomonads be recovered from the genital tract of the
heifers.

Testing the sera of 51 normally infected cows and eleven bulls,
“Organ (op.cit.) could not demonstrate the immobilization reaction.
This led him.to the conclusion that the degree of antiébody response
in the normal infection was low and I} foetus, therefore, not
highly antigenic, Thus he considered the reaction of no value as

a method for the diagnosis of Trichomonas infection. It should be

 

noted that he did not evaluate the endptiters for the simple

agglutination,as did Kerr and Robertson (l9h1).

In his work on the serological aspects of Trichomonas suis

 

sanborn (195h), employed living antigen for the inoculation of
rabbits. His agglutination procedure included a formalin - killed
antigen of a density of about two million organisms per m1, and
‘was similar to that described by Trussell (19h7). This method
allowed Sanborn (op.cit.) a distinction of the agglutination titers
of antisera, produced against different species or varieties of
the trichomonads recovered from the pig. He was able to find a

slight but definite serological difference between the two Trichomonas

 

species, one taken from.the nose and the other from.the cecum of
the pig.

Pierce (19h?) succeeded in demonstrating an agglutinin in
the vaginal discharge of four’naturally infected heifers. It was
not found in two non-infected heifers by the same technique. The
appearance of this agglutinin coincided with the disappearance of
the living trichomonads from.the vaginal or uterine discharge. The
agglutination reaction was run with a 1:10 saline- dilution of the
mucus diffusion product. There was a correlation between the de-
velOpment of the mucus-agglutinine and the serological response
of the heifers, which was demonstrable much later than the mucus
agglutination.

The local production of antibodies was confirmed by Kerr and
Hebertson (19h7). They stated that heifers having had an acute

Trichomonas infection resisted the reinfection at the time of

 

insemination and calfed at term. This was not the case with heifers

that failed to develop clinical symptoms of an infection upon the
first exposure. These authors could also demonstrate the antibodies
in the uterine secretions after an artificial sensitization of the
uterus by the instillation of the antigen. In an earlier paper,
Kerr and Robertson (19h6), had reported that the intramuscular
vaccination, in spite of a high level of circulating antibodies

and a positive skin—reaction, did not protect heifers against the
infection with I. MAntroduced vaginally at the time of
insemination.

h. Allergic skin reaction:

 

 

All of the early attempts to simplify the diagnosis of bovine
trichomoniasis by the allergic skin reaction, have been very dis-
couraging.

In l9hh, Kerr published on the intradermal test for bovine
trichomoniasis, the application of which is very similar to the
tuberculin skin test. However, it can be read after thirty minutes.
His antigen represented a complex hapten consisting of the soluble
surface component of'!. £22322, salted out with trichloracetic acid.
This test was reported to correspond well to the serological
agglutination test described earlier by Kerr and Rebertson (19b1).

5. Antigenic structure of the Trichomonas cell:

 

The recent work by menolasino and Hartman (l9Sh),throws add-

itional light on the antigenic structure of the Trichomonas cell.

 

These authors were able to separate two major components which were

effective in producing high-titer immune responses in rabbits.

Thqy also described a method of observing the antigen - antibody
reaction between the living organisms and the immune - sera, produced

against the soluble and the insoluble fractions of Trichomonas

 

vaginalis and T. foetus. Twelve to fourteen days after the last

 

injection into rabbits of either the soluble or the insoluble an-
tigen, the immune sera were harvested, inactivated and used for the
reaction with the living trichomonads.

Menolasino and Hartman (op.cit.) stated that the insoluble

antigen - component produced the fellowing types of’antibodies:

a) A specific somatic agglutinin (titer up to 1:5120)

b) A posterior flagellar'agglutinin (titer upto 1:6h0)

c) An antibody complex, causing the rounding, granulation
and fragmentation of the living trichomonads. After the
fragmentation, a flocculation of the fragments could be
observed.

They also demonstrated that the soluble antigen component

was found to produce the following types of antibodies: '

1. An antibody causing the immebilization and subsequent lysis
of the anterior flagella (up to a titer of 1:2560).

2. An antibody causing the sequence of rounding granulation
and fragmentation.

In their serological studies these authors showed that T}

vaginalis and T: foetus were antigenically indistinguishable.

However, it seemed that the two Trichomonas species could be

 

separated by their different titer to the homologous or the heter-

ologous antiserm in the cross—agglutination reactions. The introd-
uction of the agglutination of living trichomonads by Kerr and
Robertson (19hl), and the modification in the preparation of the
antigen for the inoculation of experimental animals reported by
Robertson (19h1), menolasino and Hartman (195h), seem to indicate
major progress towards a better understanding of the antigenic

behavior of trichomonads and the disease conditions caused by them.

III. MATERIALS AND METI-DDS

A. SOURCES 93 TRICHOMONAS :52.

 

The trichomonads used in this study were the same organisms

as isolated by Sanborn (1951:). TEchcmongs species (nasal isolate)

 

was obtained from the nose of a pig affected with atrophic rhinitis,
diagnosed by menbers of the Department of Animal Pathology.

Trichomonas suis, (fecal isolate), was taken from the cecum of

 

the same animal} Trichomonas gallinarum was isolated from the cecum

 

of a turkey received at the Poultry Diagnostic Laboratory at Michigan

state University. A pure culture of Tritrichomonas foetus, origin-

 

ating from the preputial washings of a bull, was kindly supplied by
Dr. Donald H. Mcflade, Dept. of Animal Pathology, Michigan state

University.

B. CULTURE MEDIA

 

For the maintenance of the Trichomonas sp. in stock, C.P.L.M.

 

medium (cysteine HCL, peptone, liver-infusion, maltose) according

to Trussell (19147), or fluid thioglycollate broth (Difco) was used.

 

«Lu

Sterile horse serum, added to both in a concentration of about four
percent, rendered an equally satisfactory growth of all organisms

in both media. In the latter part of this study only thioglycollate
broth was used in order to simplify the maintenance of stock cultures,
as C.P.L.ll. - medium was more difficult to prepare.

Another medium producing good growth of the trichomonads , and
still not interfering with the procedure of preparing the antigen
from the organism, could be found in the simple brain heart infusion
broth (BHI) without agar, to which 0.3% of’yeast extract and 0.3%
of maltose were added. Sterile, inactivated horse serum in a
concentration of about ht yielded sufficient growth for this purpose.
With eleven ml of liquid culture medium in standard (20ml) test
tubes, a 002 athmosphere as described by uenolasino and Hartman
(195h), was found to be unnecessary.

The organisms involved in this study (except I: £22222), had
been grown in artificial media for about two years,and thus had
adjusted themselves very well to cultural conditions. This was

demonstrated by the similar counts of all four Trichomonas species

 

after h8 hours incubation in the same medium.. Approximately the
same number of organisms having been employed as an inoculum.
counts made with the hemacytometer showed only insignificant
variations due possibly to the slight quantitative differences in
the inocula.

All cultures could be kept bacteria-free, but occasional
contiminants were eliminated by the addition of 1,000 units of

penicillin, and 1,000 units of

15

streptomycin per ml to the described media. This procedure did not
have any adverse effect upon the trichomonads.

C. PREPARATICN OF THE ANTIGEN

me million trichomonads were seeded into the brain heart
infusion medium specified previously. This inoculum rendered a
growth of four to eight million organisms per ml after a 148 hours
incubation at 37° C.

The test tube culture was mixed thoroughly with a sterile
lml pipette, and a sample of the trichomonads was counted in a
standard hemacytometer. Thereafter, the cells in the tube were
centrifuged for ten minutes at 1500 r.p.m., and about nine m1
of the supernatant were aseptically removed with a pipette fitted
with a rubber bulb. The settled cells were washed in sterile
physiological saline and centrifuged for another ten minutes at
1500 r.p.m. 5 this was repeated three times, the supernatant saline
being carefully discarded each time. Using sterile rubber - stoppers,
the test tubes were shaken thoroughly after each resuspension in
saline.

Next,the sediment of cells was suspended in ten ml of saline
in order to count the final number of washed trichomonads in the
bemacytometer. Usually about forty to sixty million organisms
remained in this last saline suspension. The loss during the
washing procedure seldom exceeded twenty per cent of the original

total cell count in the BHI - culture.

16

Quantities of approximately forty million washed trichomonads
per test tube were centrifuged at 1500 r.p.m. fer ten minutes.
After'drawing off the supernatant saline, the sediment was resuspen-
ded and diluted in ten ml of sterile distilled water. The tubes
were then placed in the refrigerator at h° c for three hours, being
shaken vigorously at fifteen minute intervals to prevent settling,
and to help a complete rupturing of the'Erichomonad cells as des-
cribed by monolasino and Hartman (l95h). After this three hour
period, a microscopic examination was made to check for the complete
disruption of the organisms.

In order to obtain two major fractions of this whole'nrichgmonas

 

antigen, the suspension of the ruptured cells was centrifuged at
2000 rap.m. for twenty minutes, and the clear supernatant drawn off
carefully with a sterile pipette or a syringe. This clear liquid
served as the soluble antigen fraction for the injection of the
rabbits and chickens. The dosage being measured in correlation to
_ the original number of trichomonads present before the rupturing
procedure. For example: Ten ml of the clear supernatant.repre-
seated the soluble antigen fraction of forty million living Trish:
engage cells which were broken up and lysed by the described pro-
seas.

The insoluble cell fragments remaining in the sediment of the
tube were washed and centrifuged once more in distilled water in
order to remove all of the soluble cell components. After the

supernatant of this last washing was drawn off, the sediment was

diluted with sterile saline up to the desired quantity for the in-
:jection into the experimental animals. The dosage of this milky
suspension was also correlated to the number of trichomonads pre-
sent in the test tube prior to the rupturing procedure, and was
then used for inoculation as the insoluble antigen component of
the whole Trichomonas cell.

 

Because of this rather crude method of separating two major

components of the antigen of the whole lechomonas cell, it was

 

presumed that the insoluble component still contained a minute
fraction of the soluble antigen 3 however, the clear soluble com-
ponent could be considered rather pure without containing any
"contaminating" insoluble cell substances.

D. INJECTION OF EXPERIMENTAL ANIMALS

Six female, non-prem rabbits, approximately six months old,
were selected for the first experiment. One week before the exper-
iment,each rabbit- had been bled from the heart, in order to obtain
a small amount of normal serum which, after being inactivated was
to serve as a control for the agglutination reactions.

The animals were divided into three pairs: The first pair

received the antigens derived from Trichomonas species (nasal isolate);

 

the second pair the antigen components of 11;. suis, (fecal isolate),

and the third pair was inoculated with the antigens of Trichomonas

 

gallinarum. Rabbit "a" of each pair received the soluble antigen
conponent, and rabbit "b" of each pair was inoculated with the

insoluble component of its respective Trichomonas species. Six

 

intravenous injections were made at three to four day intervals.

The animals were immobilized by rolling them tightly in a labor-
atory coat, and the injection given into the external ear-vein under
antiseptic precautions. The amount of the antigen was made up to
feur to six ml for one inoculum. The first injection represented

the antigenic components of five million living trichomonads; the
second injection equalled ten million, the third fifteen, the fourth
taenty,the fifth 25, and the sixth injection contained one of the an-
tigenic fractions of thirty million whole Trichomonas cells of the re-
spective species pertaining to each pair of the rabbits. Oh no occasion
during the injection procedures could any anaphylactic symptoms be

noticed in the experimental animals.

six white leghorn chickens, four months old, and weighing
from three to four pounds, were also divided into three pairs and
were injected into the brachial vein according to the same schedule
as the rabbits. A week prior to the beginning of this experiment,
ten ml of blood were drawn from the heart of each chicken in order
to obtain a normal serum sample as a control for the following
agglutination tests. The amount of the antigen and the time intervals
of the injections followed the pattern for the rabbits,except that
only the first four injections were made. The amount of one inocu—
lum was made not to exceed four ml per injection.

E. HARVESTING ANQ_?REPARATION OF'THE SERA

 

Ten days after the last injection in the rabbits, twenty m1

of blood were drawn aseptically from the heart of each animal.

i7

The blood was allowed to clot at room temperature, and was then
placed over night in the refrigerator at 11° C. The next day, after

breaking the clot with a sterile pipette, the blood sample was
centrifuged at 2000 r.p.m. for two hours. The serum was removed
andinactivated by heating in a water - bath at 56" c for 25 minutes,
whereupon, it was ready for the preliminary serological studies. .

Horse and cattle serum for the culture media was prepared in
much the same manner, except that it was collected in larger quan-
tities from the jugular vein of these animals.

In order to maintain the level of antibodies in the sera, one
week after the first bleeding of the rabbits, all animals were
reinjected with their appropriate antigen, corresponding to forty
million living trichomonads. Four days later the same amount was
again injected. Six days after that all rabbits were bled out in
the same marmer as described, and sacrificed.

The chickens were bled from the heart one week after the last
inoculation, and the sera harvested in like manner as the rabbit
sera. No challenging reinjection was applied.

F. AGGLUTINATIGW REACTICNS

The agglutination reactions were set up in test tube racks,
containing ten (12 x 100 mun) serological tubes. To each tube, 0.5
m1 of sterile physiological saline was added. The lowest dilution
of the antiserum was made up in an extra tube as follows: 0.2 ml
of the serum was diluted with 3.8 ml of saline, thus making a 1:20

dilution. From this tube serial dilutions were made down the line,

20

using 0.5 m1 transfers. The senmnvsaline dilution of the first

tube was mixed thoroughly with a lml total delivery pipette, and

a clean pipette was used for each subsequent serial transfer through
tube number nine. Tube number ten served as the saline control for
the antigen. For the antisera, dilutions ranging from 1:110 (tube
number one), to l: 5120 (tube number nine) were made; in the case

of the normal rabbit and chicken sera dilutions were set up from
1:5 up to 1:6h0, adding 0.2 ml of serum to 0.8 ml of saline in the
first tube.

Beginning with tube number ten, a 1:1 ratio of the living
antigen was added to the saline control and the serum-“line mixtures J
this meant onenl being added to tube number nine and 0.5 ml to all
other tubes. The density of the antigen had been adjusted to
500.000 trichommads per ml. This could generally be accomplished
by a 1:10 saline dilution of a brain heart infusion culture, having
been incubated at 37° 0 for us hours. Living antigen prepared by
this convenient method of direct saline dilution showed equally
good results as did an antigen of the same density, washed comp-
letely free from the culture medium.

The entire rack of tubes was shaken gently and placed into a
32° 0 water bath. This temperature was found to interfere less with
the viability of the antigen in the saline controls, than did 37° c.

After thirty minutes incubation the first reading of the
agglutination was made by microscOpic obersavation. A 25 x 75 mm

slide was divided into ten squares with a wax-pencil. By means

\

of a capillary pipette; a small drop of the saline control and the
serum - antigen mixturth (approximately 0.015 ml),was drawn from
the bottom of each tube, and placed in the proper square of the slide.
The drops were allowed to remain for about 3 to 5 minutes at room
temperature and were then quickly examined under the low power of
a standard microscope for the different types of agglutination. A
slide was discarded after 15 minutes due to interference by the air
oxygen in the saline control and in the serum. dilutions. There-
fore, the incubation of the slides for thirty minutes in the moist
chamber (instead of the test tube - water bath incubation) was
considered as unreliable.

Further readings by the same procedure were made after orehour,
two hours, three hours, six hours, twelve hours, and 2h hours
incubation. Care had to be taken that the small portion of the
antigen-antiserum mixture , to be examined ,was taken from the bottom
of the tubes in order to get the highest concentration of trichomonads
on the slide. This could best'be accomplished by drawing a two to
three inch liquid column from the bottom of a tube with a capillary
pipette and expelling about two thirds of this column. A drop of
the remaining fluid was then placed on the proper square of the slide.
It was necessary to handle the test tube racks carefully to prevent
undue agitation.

The organisms showed the tendency to assemble in the center
of the drop on the slide. The agglutination reactions were read

serially beginning with the saline control and continuing through

the highest dilution. First, the degree of motility of the living
antigen was estimated and recorded. If the motiliiy of the trich-
omonads present in the saline control was less than fifty percent,
the whole test-rack was discarded. This only happened at times when
older antigen was used or when the temperature of the water bath
rose too high. While observing the agglutination of the living
antigm, fresh agglutinations had to be differentiated from agglo-
merating dead, degenerated, or partly dissolved cells, usually
found together with cellular debris, and sometimes encountered also
in the saline controls. More than two freshly agglutinated units
per microscopic field,each involving at least two cells, were
recorded as "one plus" ( + ). If approximately } of the trich-
omonads in a field were agglutinated, the reaction was recorded as
"two plus"; if about i of the cells were agglutinated, the reaction
was considered as "three plus", and when more than 3/1: of the org-
anisms were involved in agglutinations, the result was noted as
"four plus" .3

G. DIFFERENTIATICN OF THE TYPE OF AGGIJJTINATION

Since two basically different types of agglutinations were
encountered corresponding to the anti-sera produced by the two

different components of the Trichomonas antigen, it was felt

 

necessary to describe these phenomena accordingly:
1.) Groups of trichomonads were seen held together by their
posterior or trailing flagella, moving their bodies vigorously

without necessarily showing a bodily contact. Round and symetrical

rosettes of’moving trichomonads could generally be noticed in this
type of agglutination which was designated as "posterior flagellar
agglutination" (PFA). The organisms showed.no tendency to per-
manently adhere to their bodies or anterior flagella. Thus they
were able to move vigorously, in most instances displaying desperate
efforts to escape centrifugally from their centerpoint of attach-
ement. In small rosettes, encountered in the higher dilutions of
the antisera, this centerpoint was found to be the very distal part
of their posterior flagella.

In the lower dilutions of the respective anti-seragup to several
hundred organisms could be observed in ball-like clumps, constantly
moving on the periphery, and thus somewhat resembling a colony of
Volvox globator (Figure l).

2.) The second type of agglutination was represented by small
or large groups of trichomonads adhering laterally. This intimate
adherence did not permit the organisms to more as in the case of the
posterior flagellar agglutination. These cellular agglutinations
were found to occur in very irregular shapes and never could typically
round balls or rosettes be seen. This type of agglutination was
therefore designated as "somatic", or "0" - agglutination (GA),
(Figure II),only a very few of the peripherally attached cells were
showing some movement of their anterior flagella. The intensity of
the reaction in the serial dilutions was likewise also recorded from
"Doubtful" ( 1'. ) to N four plus", as in the case of the posterior

flagellar agglutination. It was also found necessary to record

the degree of imbililation of the living antigen, occurring in the
lower dilutions of this type of agglutination.

H. CROSS AGGLUTINATIONS

Besides the regular, homologous agglutination reaction (i.e.
each antiserum produced to the respective antigenic component of
one [richomonas - species was reacted with the living antigen of this

 

same organism), heterologous agglutination. tests were also conducted
by testing each antiserum with the living antigen of all four Trich-

omonas species involved in this study.

Figure l.

POSTERIOH FLAGELLAH AGGLUTINATION (P.F.A.)

 

Figure 2.

SOMATIC AGGLUTINATION (0.A.)

 

1) “STE I e

r i. ,0"
'f 2.;
U .u‘ 'g
f
h \“ II.
'» e
'r
. h '
:10“ 'v __ .
. . " '. ' . .‘ :
e . a q 9‘ - -.
" e+ * a ‘ ' '
9 ' . , \ I. .e
e _ u . aka 0
e k a .I K ..
\v H ' ,- 0'
‘ {I ‘
N .
s ' ~‘ 3";
e

 

Somatic agglutination (O.a.)
ca. 100 x

 

Posterior flagellar agglutination (P.F.A.)
ca. 600 x

L)

IV. RESULTS

A. NORMAL SERUM AGGLUTINATIONS

1- rel-in em.

The inactivated serum controls of all rabbits, involved in the
study, showed normal agghtiriaztion titers for 1;. species, (nasal isolate)
I. g!) (_i_‘_e__ca_]_._ isolate), and 2. gallinarum, varying from 1:10 to
1:140. Readings'were made after thirty unnutes andcrs hour. It was
found that the type of this normal egglutination was predominantly
a somatic agglutination. mly the highest dilutions showed some
typical P.F.A., and this mainly at the thirty minute reading. A

differentiation of the three Trichomonas species according to

 

their normal titers was not attempted.

2. chicken serum:

 

The normal agglutination titers of the chicken sera were found
to lie between 1:20 and 1:10, as read after thirty minutes and one
hour. P.F.A. was predominating in the higher dilutions; only
the lower dilutions showed a predominating "0" agglutination of
the trichomonads.

3. Horse serum:

 

Normal inactivated horse serum reacted with the living antigen

of the two Trichomonas species from the hog, E. gillinarum, and

 

 

1. foetus, in a titer from 1:320 to 1:610. The type of agglu-
tination was predominantly a P.F.A.,like in the case of the normal

chicken sera. No significant difference in titer could be found

26

in the reactions to the different Irichomongg species.

14. Cattle serum:

 

‘i'he inactivated sera of three virgin heifers, reacted with g.
m showed a titer varying from 1:80 to 1:320 (average 1:160),
as read after thirty minutes and one hour, with P.P.A. predominating
in the higher dilutions and 0.1, in the lower dilutions. 89ml,
inactivated beef serum used in the later part of the culturing work,
had an average titer of 1:160,” reacted with the different £333.}:
m species.

homered to the results obtained by lax-gas (19%) with 1', foetus,
the norm]. titers of the rabbits were'htgier (1:10 to like: lorgan
1:10; also the titers of the chicken sera were slightly above the
results of Iorgan (1:20 to 1:10: uni-gen 1:16). Normal titers of
horse sera used for culturing work were found to be considerably
lower "than the titers observed by florgan for 2.2 £2221 (1:320 to
1:510; “organ 1:102h, {5+er ). the normal levels for 3:. M
obtained frm the heifer and from the cow sera cospared closely
with the findings of Morgan (1:160, norgen 1:128),

‘SB. ANTISERU'M AGGLUIINATIWS AN D CFDSS AGGLUTINATIONS.

1e m..mt186rls

‘) Antiserum produch against the. soluble cell substances. of
3. species, (nasal isolate), , reacted, with, the homologous living
antigen of the same species, and with the heterologous living antigens

of g. suis, (fecal isolate), LI". gallinarum, and l. foetus. (Table ;l)

27

Explanation of Il‘ahles I through XIII:
P. F.A. a posterior flagellar agglutination only
OJ. = somatic agglutination only
(P.F.A., 0.1.) x P.F.A. predominating over 0.A.
(O.A., P.F.A. )= 0.1. predominating over P.F.A.

+ a: two or more freshly agglutinated units per microscopic field
+ + ,7; approximately } of the trichomonads involved in agglutination
+++ -_.- approximately it of the trichomonads involved

++++ = more than 3/1.; of the trichomonads agglutinated

The titers given represent the readings after 30 annutes and
one hour. A animus of three tests for each single reaction was

performed in the same manner.

TABLE I
01-088 THRATION OF THE ANTISERUI P301110-
AGAINST THE SOLUBLE CELL FRACTION OF TRICI-DIIGJAS SP.
(NASAL ISOLATE).

 

 

Living antigen of {titers
at '_r_.sp. (nasal isolate) 1:1280 P.F.A.
I. suis (fecal isolate) 1:61.10 _P.F.A.
11:. gallinarum 1:320 P.F.A.
g. foetus 1:320 P.F.A.

 

*- homologous reaction

b) Antiserum produced against the insoluble cell substances
of _'1'_. species, (nasal isolate) reacted with the homologous living
antigen of the same species, and the heterologous living antigens
of _'1_'. £11, (fecal isolate), 1. gallinarum, and-'5. foems.

 

TABLE II
(moss TITRATION OF rm; musm PRODUCED
mum THE msomms cm. monons or
TRICPDMGIAS SP. (NASH. ISOLATE)

 

* titers
Living antigen of Highest dilution next lower
- dilution

Vfi

«- ‘1'. species (nasal isolate) , .lzg560(O.A.,P.l'.A.) 1:1280 (O.A.)

I. suis (fecal isolate) 136110 (O.A.,P.F.A.) 1:320 (O.A.)
2:. gallinarum 1:6110 (O.A.,P.F.A.) 1:320 (O.A.)
Is fat“. 126110 (00‘s)

a homologous reaction

 

o) Antiserum produced against the soluble cell substances
of 2. suis, (fecal isolate), reacted with the homologous, living
antigen of the same species, and with the heterologous living anti-

gens of ‘_1'_. species, (nasal isolate), I. allinarum, and 1. foetus.

TABLE III
CROSS TITRATION OF THE ANTISEW mecm AGAINST
THE SOLUBLE CELL SUBSTANCES OF I. SUIS (FECAL ISOLATE)

 

 

Living antigen of titers

a 2. suis (fecal isolate) 1:6h0 P.P.A.
2. species (nasal isolate) 1:320 P.F.A.
I. fill-mm 1:320 Pep-As
'1'. foetus- 1:320 P.P.A.

 

a homologous reaction

d) Antiserum produced against the insoluble cell substances
of _'1_'_. 211:, (fecal isolate), reacted with the homologous living an-
tigen of '_r_. £1_s_,(fecal isolate), and with the heterologous living
antigens of '_I'_. species, (nasal isolate), I. gallinarum, and f1_‘. mug.
TABLE IV
CROSS TITRATIQI OF THE ANTISERUH PRDWCED AGAINST
THE DISOLUBLE CELL FRACTIONS 0F 11:. §_U_l_S_ (FECAL ISOLATE)

 

 

living antigen of titers

«- 1. suis, (fecal isolate) 1:6b0 (0.A.)
2. species, (nasal isolate) 1:320 (0.A., P.F.A.)
g. gallinarum 1:320 (0.A., P.F.A.)
I. foetus 1:320 (0.A.)

 

* homologous reaction

e) Antiserum produced against the soluble cell substances
of 2. gallinarum, reacted with the homologous living antigen of the
same species, and the heterologous living antigens of 2. species,

(nasal isolate), Z. suis, (fecal isolate), 11:. gallinarum, and g.

 

 

 

 

foetus _
TABLE V
CROSS TITRATICN OF THE ANTISERUII PRODIL‘ED AGAINST
THE SOLUBLE CELL SUBSTANCES OF I. GALLINARUII

living antigens of titers

a Z; gallinarum 1:1280 P.F.A.
2. species, (nasal isolate) 1:61:0 P.F.A.
g. suis, (fecal isolate) 1:6h0 P.F.A.
Z. foetus 1:320 P.F.A.

 

a homologous reaction

1‘) Antiserum produced against the insoluble cell fractions
of I. gallinarum, reacted with the homologous living antigen of the
same species, and with the heterologous living antigens of ‘1'. species,

(nasal isolate), 2. suis, (fecal isolate), and I. foetus.

d) Chicken antiserum produced against the insoluble cell

fractions of 1. suis, (fecal isolate), reacted with the living

 

antigen of the four Trichomonas species.

TABLE II
CROSS TITRATION OF THE ANTISERUH PRODUCED AGAINST

THE INSOIUBLE CELL FRACTICNS OF 2. SUIS (F'ECAL ISOLATE)

 

titers
living antigen of highest dilution next lower dilutions

LA

«- 2. suis, (fecal isolate) 1:1280 (P.F.A.0.A.) 106140 ( 0.A.P.F.A.)
2. species. (nasal isolate) 1:320 (P.F.A.0.A.) 1:160 ( 0.A.P.F.A.)

1. gallinarum 1:320 (0.A.P.F.A.)
20 room 1:320 (OeAe)

a homologous react ion

 

e) chicken antiserum produced against the soluble cell

substances of 2. gallinarum, reacted with the living antigens of the

 

four Trichomonas species:

 

TABLE XII
CROSS TITRATION OF THE ANTISERUM PRODUCED AGAINST THE

SOLUBLE CELL SUBSTANCES OF I. GALLINARUM

 

 

 

Living antigen of titers

of. gallinarum 1:1280 riff.
2. species, (nasal isolate) 1:320 P.F.A.
2. suis, (fecal isolate) 1:6h0 P.F.A.

'r. foetus 1:340 P.F.A.

L-....‘I -nme- mos§§ nn

 

2. chicken Antisera

 

a) chicken antiserum produced against the soluble cell frac-
tione of 1. species, (nasal isolate), reacted with the living antigens

of the four different Trichomonas species.

 

TABLE VIII
CROSS TITRATION OF ANTISERUM PRODUCED AGAINST TIE
SOLUBLE CELL FRACTIONS 0F 1. SPECIES (NASAL ISOLATE)

 

living antigen of titers

a _'1‘_. species (nasal isolate) 1:1280 P.F.A.
2. suis, (fecal isolate) 1:320 P.F.A.
g. gallinanm 1:320 P.F.A.
2. foetus ‘ 1:160 P.F.A.

* homologous reaction

b) chicken antiserum produced against the insoluble cell
fractions of 11;. species, (nasal isolate), reacted with the living

antigens of the four Trichomonas species.

 

0. SOME NOTES ON THE GROWTH RATES AND THE LONGEVITY 0F Lam-
w SP. IN DIFFERENT CULTURE MEDIA.
1. Growth Rates
Though this study cannot contribute very much to the laborious
work of culturing trichomonads done by many. research workers, it
was felt important to include some data and minor observatiors which
may help future research, in order to obtain a more satisfactory

medium for the diagosis of Trichomonas : diseases.

 

All the culture media described in Materials and Methods, rend-
ered a good growth of the trichomonads; using one million organimns .
as an inoculum into 11 m] of media with 1:8 hours incubation at
37° C, the highest concentration of the trichomonads was obtained
in the C.P.L.M. - medium, followed by thioglycollate broth and the
modified BBL-medium.

It was found that the mount of serum (10% recommended by
Trussell, 191:7; 20% recommended by Menolasino and Hartman, 19514)
could be lowerd to hi without adverse effects on the cultural work
to be done. Also a carbon dioxyde atmosphere of 10%, as reported
by Menolasino and Hartman (19511), could easily be omitted.

Due to the relatively high normal titer of horse serum for all
Trichomonas sp., beef serum proved even more advantageous and
rendered equally high or higher counts under the same conditions.

The following average counts per ml could be determined after
1:8 hours incubation at BIO c, with an initial inoculum of one million

organisms into llml of medium:

Eacplanation for tables XIV aadXV:

Numbers represent millions per ml

B.H.I.Y.M.S.A. _._ brain-heart infusiomplus 0.3% yeast extract,
plus 0.3% maltose, plus h % horse serum,plus antibiotics.

Thio 3A,: Brewor's thioglycollate broth,plus 14% horse serum,
plus antibiotics.

TABLE XIV

AVERAGE COUNT IN MILLIONS PER Ill AFTER ’48 HOURS INCUBATIGQ

;AA

Trichomonas sp.
medium T. species, T. suis, T. gallinarum T. foetus
'(nasal (fecEI " "‘
isolate) isolate)

 

 

 

B.H.I.I.I.3.A. 7.01 7.b6 6.81 5.77
(30 counts)
T1110. S.A. 70,49 Bell 8e60 7am
(20 counts)
TABLE IV

AVERAGE COUNTS IN MILLIONS PER I1 AFTER 72 HOURS INCUBATION

 

 

 

Medium Trichomonas sp.
B.H.I.Y.M.S.A. T. species T. suis I. gallinarum 1. foetus
(10 counts) "(nasal (fecal:

isolate) isolate)

5.76 b.28 3.71 5.01

 

The speed of growth was fastest in the thioglycollate and in
the C.P.L.ll. medium, and slower in the modified B.H.I. medium. The
peaks in growth were usually reached after 36 to 148 hours incubation.
After that time there followed a quick dying off process of the
trichomonads, which was most pronounced in the thioglycollate broth,
and less in the modified 3.11.1.3 C.P.L.M. medium standing in
between.

All media had an initial pH of 7.0. As reported by Trussell
(19M). and confirmed by Sanborn (195k), the «mug of the organism
in the media went hand in hand with a lowering of the pH. This
led Russell (op.cit.) to the conclusion that the trichomonads are
killed by the acid production in the medium. The observation of
Sanborn (op.cit.) could be confirmed in this study, that the acid
production by the trichomonads did not have a primary effect on the
killing of the organisms, though it showed a definite correlation
with the dying rate of the trichomonads in vitro. A 63-day old
C.P.L.M. medium with a pH of 13.5 and 5.0 in one instance, rendered
the same growth rates as did fresh medium with a neutral pH.

2. OBSERVATIONS ON THE LONGEVITY OF THE TRICHOMONAS SP.

 

It was reported by Switzer (1951), and Sanborn (1951:), that

Trichomonas cultures lasted longer if kept at room temperature.

 

Sanborn (op.cit.) stated,that C.P.L.M. cultures kept for five to
seven days at 37° C incbation. In the present study this was also
found to be true for the modified B.H.I. medium, while in the

thioglycollate broth after 5 days at 37° 0 usually no living

organisms could be found. According to Sanborn (195M, thiogly-
collate and B.H.I. medium did not support the growth of the trich-
omonads from the hog, or I. foetus. In thioglycollate (without
serum?) , he found them alive for two to three days without unllti-
plication; in B.H.I. semisolid (without serum), the orgaisms
were reported to stay alive for about fifteen days without multi-
plication. In this work, however, the hog trichomonads and 1.
gallinarum could be kept alive in fluid thioglycollate medium with-

 

out serum for at least 7 days at 37° 0. Using an inoculum of
10.000 trichomonads per ml, there was a thirty to forty fold
multiplication after he hrs 37° c. For fluid B.H.I. with 0.3%
yeast extract, but without serum or agar, the multiplication factor
was three to five fold after the same time.

In longevity tests at room temperature with the complete media,
containing about 14% of horse serum, all of these trichomonads could
routinely be kept for about ten days in thioglycollate and B.H.I.
medium after an initial incubation of 214 hours at 37° c. In
C.P.L.ll. medium the organisms could be found alive after 23 days.
This compares well with the results obtained by Sanborn (19514). On
one occasion 1. species. (nasal isolate) was still living after 32
days in C.P.L.M. medium.

In high test-tubes (15 x 200mm), with a liquid colum of the

medium of about 11:0 mm (18ml volume), 2. gallinarum and the two

 

hog Trichomonas sp. kept mobile for about thirty days in the fluid

 

B.H.I. medium with 11 % horse serum. The same Trichomonas sp.

 

were positive after 60 days in the thioglycollate medium (plus 14%
seam), and :11. gallinarum and 1. species, (nasal isolate) were
found to be still alive after 103 days under the same conditions.

In an accidental finding, 2. species, (nasal isolate) showed
motile trichomonads after having been stored in the ice box at 14° 0
for h2 me. In another instance,the same species was found alive in
B.H.I. sousolid (hi ser1m) after a storage of fifty days at room
temperature.

 

f”

a,

 

V
DISCUSSION
Since this study was dealing with well adapted, bro year-old
laboratory cultures (except 1. m), the minor differences in
growth rates, speed of growth and longevity were not thought to be
significant enough to characterize a separation of the different
Trichomonas sp. on the basis of these findings.

 

Though the serological procedures were modified and different
from those of previous workers, the titers obtained especially in
the homologous reactions compared closely to the earlier reports.

As there is no standard procedure for living Trichomonas

 

agglutinations worked out at the present tile, the results of diff-
erent workers have to be evaluated accordingly. Also the human
factor in Judging the titers must be considered.

It was felt that the establishment of an agglutination titer
for normal or immune sera of domestic animals with a living protozoan

like Trichomonas is a process which differs in some respects from

 

the agglutination of fomlinised bacteria. In observing the slide-
agglutinations and in checking the reactions for an extended time,
biological and physical factors are liable to cloud the picture,and
lead to wrong interpretations. The agglutination incubation time
was therefore standardized to not more than two hours and the results
of later readings were not evaluated in this work.

Further research is necessary to study the degeneration proc-

esses under the adverse environmental conditions created by the

agglutination procedures. A more thorough knowledge about the
phenomenon of spontaneous agglutination would be desirable. This
phenomenon was occasionally seen under the microscope in samples
from culture media as well as in the saline controls,after having
been exposed to the air for a certain time. Because of its close
resemblance to the.P.F.A., special care had to be taken in the
reading of this type of agglutination. I

contrary to the report of Sanborn (19Sh), that there was: l'Il.ittle

 

or*no evidence of a group antigen among the two Trichomonas species
from the hog and.!} foetusz " the results of this study showed a

strong evidence of a common group antigen among all four Trichomonas

 

sp. used. The present isolate of'!} foetus, which was not the same
culture as used.by Sanborn (op.cit.), was acting as a control.

This points out the antigenic similarity of all Trichomonas species

 

involved.

‘While uenolasino and Hartman (l95h), in similar studies on T.
foetus and.T: vaginalis came to the conclusion that these two org-
anisms were antigenically indistinguishable, Sanborn (l95h), stated
that :"The agglutination experiments showed conclusively a definite

antigenic difference between the two Trichomonas forms used from

 

the pig.”

According to the results of this work neither of the foregoing
statements can.be agreed upon completely.

For both antisera produced against the soluble and insoluble

antigen fractions,the cross agglutination titers between the two

hog Trichomonas forum compared to the findings of Sanborn (op.cit.),

 

though the differences in titer were not as striking as in Sanborns
work. However, it was felt that these differences were significant
and consistent enough to Justify a differentiation of the two W
cm species from the pig, as stated by Sanborn (op.cit.).

It was also found that the titers obtained from the homologous

and heterologous reactions of all Trichomonas sp. studied, resembled

 

closely the titers given by Ilenelasino and Harman (1951;), for the
somatic agglutination of 11:. m and 11;. v_s_.ginalis.

All the culture media used in this study (C.P.L.ll.,
thioglycollate, B.H.I. modified, and B.H.I. semisolid), rendered
satisfactory growth rates. This was somewhat in contradiction to
the results of Sanborn (19514), who reported that thioglycollate
and B.H.I. did not support the growth of his Trichomona_s_ sp.

 

Continuous transfers of the trichomonads were possible in all
of the media described above. The serum content in the media could
be lowered to. 13% without any detrimental effects. Special precautions
like a 10% 002 athmosphere according to Honolasino and Hartman (1951;)
were found to be superfluous.

The average counts after 1.8 hours incubation are self explanatory
and lie above the averages reported by Smborn (1951;); about one
million organisms were used for the initial inocula per test tube,
in comparison to Sanbom (op.cit.), who obtained the best results

with inocula of 100,000 to 200,000 into 10 m1 of medium.

11.! l.i..lv\11

Experiments with the longevity of the trichomonads were not
routinely performed since this was not a primary purpose of this work.
Here data would be necessary to confirm the findings. The results

'were added to demonstrate that Trichomonas in general is not as

 

fastidious an organism.as was thought.previously, and that the
artificial culturing and the maintenance of this protozoan can still

be simplified.

 

VI
SUMMARY AND CONCLUSIONS

The serological behavior of two Trichomonas species from the

 

pig,as well as 1.2. gglinarum, aid 1. mp“ studied in this
work.

A procedure for the living agglutination reaction of trich-
omonads was described and interpreted. This procedure utilized a
modified antigen for the immunisation of eXperimental animale,and
employed a different method of observing (reading) the agglutination
of the living organisms under the microscope.

Antisera produced against the two major fractions of the
Trichomonas cell resulted in two different types of agglutination:

1. If reacted with the antiserum.produced against the waterb

soluble antigen fraction, all four Trichomonas species

 

showed a typical posterior flagellar agglutination.

2. Antiserum produced against the water extracted ("insoluble")
antigen fraction resulted in a typical somatic agglutination

of all four Trichomonas species, and in some instances a

 

somatic and a posterior flagellar agglutination occurred

simultaneously to a lesser extent.

3. The pooling of both kinds of antisera produced both types
of agglutinations, with the P.F.A. predominating in the
higher dilutions and the 0.A. predominating in the lower

dilutions .

 

‘4)

cross titration studies with the homologous and the heterologous
living antigens showed a strong evidence of a cannon group antigen

in'all four Trichomonas species. The isolate of l. foetus acted as

 

a control. However, it was noted that the agglutination titers of
the homologous reactions were consistently higher than the titers
of the heterologous reactions. This difference in the cross-titrations
was thought to be significant enough to Justify a differentiation
between the trichomonads used in this study, and especially, be-
twaen the two species isolated from the nose and from the cecum of
the pig, as reported by Sanborn (19514).

The use of ruptured trichomonads as an antigen for the
immization of experimental animals seemed to be more successful
in eliciting a high antibody response ,than was the application of
the living or formalinized,intact trichomonad antigen reported by
earlier workers. (Sanborn, 1951:; Horgan, 191:3, 19146; Trussell, 19116,
19147).

According to the results of the serological reactions confirmation

was obtained that, like _'1_‘. foetus and g. vaginalis as reported by

 

Menolasino and Hartman (1951:), also the two pig trichomonad species

and I. gallinarum are highly antigenic.

 

Cultural studies with the media employed proved highly satis-

factory, and simplified procedures could successfully be applied
for all trichomonad species used in this work.

VII

BIBLIOGRAPHY

*Abelein, R. 1929. Beitrag zur Kenntnis des von der Banginfection
unabha’ngigen whom»
lunchener Tierzrztl. lochenschr. 80: 637-675.

*Byme, J. 19h2. Trichomonas foetus; Infection and immity in

 

 

rabbits. Thesis, Univ. Wis. 33 nunb. leaves.

*Donne, A. 1836. Animalcules observes dans les matibres purulentes
et le produit dos secretions des organes genitaux de
l'home et de la fem. compt. Rend. Acad. Sc. 3:385-386.

Summon, ILA. 1932. Trichomoniasis in cattle. Jour. Amer. Vet.
lied. Assoc. (81) 3b: 636-6140.

*Endress, R. 1939. Verwertbarkeit der Komlementbindungsreaktion,
der Agglomeration und der 'rrichomolyse far die Erkennung
der 'rrichomonadeninfektion des Rindes.

Arch. 1. Wiesensch. u. Prakt. Tierheilk. 75: 65-82.
Hayes, F. A. 1955. Oral commmication. Univ. of Ga. School of
Vet. Med.

*Hoehne, 0. 1916. Die Behandlung der g‘_1:ichomonas Kolpitis. centralbl.

 

Fa 03112.1(01. 1403113‘118.
*Ibid. 1916. Trichomonas vaginalis als hzufiger Erreger einer

 

typischen colpitis purulenta. Centralbl. F. Gyngkol.
’40: 11.150

* original articles not seen

Johnson, 6., and Trussell, R.E. 191:3. Experimental basis for the

chemotherapy of Trichomonas vaginalis infestations.

 

Proc. Soc. Exper. Biol. and Med. Shz2h5-2h9.

Kerr, I. R. 19141:. The intradermal test in bovine trichomeniasis.
vet. Rec. 56: 303-307.

Kerr, ER. , and Robertson, II. 19111. An investigation into the
infection of cows with Trichomonas foetus by means of
the agglutination reaction. Vet. Jour. 97:351-365.

Ibid. 19146. Experimental infections in virgin heifers. with
Trichomonas foetus in vaccinated and unvaccinated
animals. J. Comp. Pathol. 56: 101-113.

Ibid. 191:7. A stuck of the reexpesure to :11. foetus of animals
already exposed to the infection as virgin heifers, with
some observations on the localisation of antibodies in
the genital tract. J. Comp. Pathol. 57:301-313.

*Kunstler, J. 1888. Surquelques infusoires nouveaux eu peux
' connues. Compt. Bend. Acad. Sc. 107:953-955.
*Mazzanti, B. 1900. Due osservazioni zooparassitologiche. Gior.
12., Soc. ed Accad. Vet.Ita1. [19:626-631.
Menolasino, N.J., and Hartman, E. 19514. Imunology and Serolog of

some parasitic protozoan flagellatee I: Trichomonas

 

vagi_nalis and Trichomonas foetus. J. Immol. 72:172-176.

 

Mbrgan, 3.3. 19b3. Studies on the immdbilisation reaction of
Trichomonas foetus (Protozoa) in cattle. J. Immol.
’47: 1453-4460.

a original article not seen.

 

Ibid. 19hh. Normal agglutinine in vertebrate sera for Trichomonas

 

foetus (Protozoa). Proc. Helminth. soc. Washington 11:21-23.
Ibid. l9h6. Bovine Trichomoniasis. Burgess Publ. Co. Minneapolis, l65pp.
Morgan, B.B.,and Beach, B.A. 19h2. The geographical distribution of
bovine trichomoniasis. vet. Med. 37: h59-h62.
emerisita, T. 1939. Studies on the trichomonad, parasitic in the
reproductive organs of cattle. Jap. J.‘EXper. Med. 17:1-62.
Nelson, P.M. 1937. Antibodies for Trichomonas foetus. Arch. Pathol.
23:7hh-7b5.

 

Ibid. 1938. Trichomonas foetus; Infection, immunity, and chemotherapy.

 

Thesis, Univ.‘Wisconsin, 18pp.

*Pfenninger, W. 1927. Waiters Beobachtungen beim.sporadischen
Abortus des Rindes. Manchener Tiergrztl.‘Wochenschr.
78:217-219.

Pierce, A.E. 19h7. The demonstration of an agglutinin to Trich-

 

omonas foetus in the vaginal discharge of infected heifers.
J. Comp. Pathol. 57:8h-97.

Riedmfiller, L. 1928. fiber die Morphologie, fibertragungsversuche
und.klinische Bedeutung der beim sporadischen Abortus
des Rindes vorkommenden Trichomonaden. Zentralbl. f.‘Bact.
I. Abt. Orig. 108:103-118.

*Ibid. 1932. zur Frags der gtiologischen Bedeutung der bei Ryometra

und snoradischem Abortus des Rindes gefundenen Trichomonaden.

Schweiz. Arch. F. Tierheilk. 7h:3h3-351.

* original articles not seen

* Rivolta, S. 1878. Una forms di croup prodotta da un infusorio, nel
polli. Giorn Anat. Fisiol. e Pathol. Anim. 10:1h9—158.
Robertson, M. 19hl. Agglutination reactions of certain trichomonads
in sera obtained from.immunized rabbits, with particular

reference to Trichomonas foetus. Jour. Pathol. and

 

Bacteriol. 53: 391-h02.

Sanborn,‘W.R. 195k. studies on cultural, serological, and other
aspects of trichomonads associated with atrophic rhinitis
and similar studies on other trichomonads. Thesis, Mich.
State College, 53 numb. leaves.

Ibid. 1955. Microagglutination reactions of Trichomonas suis,

 

 

2} sp., and I, foetus. Jour. Parasit. hlz295-298.

Schneider, M.D. 19hl. Trichomonas foetus in cattle with special

 

reference to diagnosis. Thesis, Univ. Wisconsin, 33 numb.
leaves.
Ibid. 19h2. A new thermostable medium for the prolonged bacteria -

free cultivation of Trichomonas foetus. Jour. Parasit.

 

283h28-b290
Switzer,‘W.P. 1951. Atrophic rhinitis and trichomonads. Vet. Med.

stabler, R.M. 195h. Trichomonas gallinae: A review. Exper. Parasit.

 

3:368-h02.

Traits de zoologie, Tom. I. Masson et cie., Paris 1952. 1071 pp.
1952.

* original articles not seen

Trussell, 11.13:. 19146. Microagglutination tests with Trichomonas

 

vaginalis. J. Parasit. 32: 563-569.
Ibid. 19147. Trichomonas Vaginalis and Trichomoniasis. charles c.

 

'Thomas, Springfield, :11. first ed. 277pp.
*Witte, J. 193k a. Infektions versuche an Rindern zur kLHrung
der pathogenen Bedeutung der Trichomonaden far die sterilitgt
und-dsn Frahabort dos Rindes. Berliner Tierzrzf1.'WOchenschr.
50:56h-566.
*Ibid. 1931: b. Serologische Untersuchungen zum Nachweis der Trichom-

onadeninfection der Genitatien dos Rindes. Berliner
Tiergrztl. Wochenschr. 50:693-695.

*Zeeti, R. 19h0. Ricerche sperimentali sul Trichomonas flMhzzanti."

 

La Clinics Vet. (Milano) 63:5-16: 51-59.

* original articles not seen

SEROLOGICAL AND IMMUNOLOGICAL STUDIES

ON SEVERAL ANIMAL TRICHOMONAS SPECIES

 

BY

Hans Jacob Rothenbncher

AN ABSTRACT

Submitted to the School of Graduate Studies of
Michigan State university of Agriculture and
Applied Science in partial fulfillment of the

requirements for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

year 1955

Approved by

 

With a modified procedure for the trichomonad living agglutination

the serological behavior of three Trichomonas species was studied. For

 

the production of antisera in rabbits and chicken,the trichomonads were
ruptured.and the antigen applied in two major fractions by intravenous
injections.

Serological tests conducted with the antisera and the living
antigens revealed two different types of agglutination according
to the antigenic fraction of the trichomonad cell used for the immun-

ization of the animals.
Antisera produced against the water soluble fraction of the

Trichomonas cell resulted in a typical posterior flagellar agglutination,

 

while the antisera produced against the water insoluble fractions
created a typical somatic agglutination of the trichomonads, as seen
under the microscOpe. The pooling of both kinds of antisera produced
both types of agglutination simultaneouslyywith the P.F.A. predom-
inating in the higher dilutions,and the 0.A. predominating in the
lower dilutions.

Motives for conducting these agglutination tests were: to clarify
whether this method was applicable for the specific differentiation
of the trichomonads, and secondly, to work towards the deve10pment of
a practical testing procedure which might be useful for the diagnosis

of important Trichomonas diseases.

 

In cross titration studies using the living antigens of Trich-

omonas sp. (nose of the pig), 2? suis(cecum of pig), 2, gallinarum,

 

and T. foetus, a common group antigen of these species could be

noticed. It was also noticed that the titers with the homologous

antisera were consistently higher than those of the heterologous
reactions. This difference was thought to be significant to Justify
a differmtiation of the trichomonads involved in this study, and

particularly, of the two Trichomonas species isolated from the pig.

 

contrary to reports of earlier workers (Morgan, 19113; Sanborn
1951;), the observations made by Menolasino and Hartman (19511) , that
1. vaginalis and 1. foetus are highly antigenic, could fully be

confirmed also for the two Trichomonas species from the pig, and .‘I_'.

 

gallinarum.

In cultural studies with different media, it was found that
simplified procedures could be applied for the trichomonads without
detrimental effects. It was felt that Trichomonas in general is not

as fastidious an organism as was thought previously.

 

 

 

 

 

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