eScholarShare at Drake University

Fors, Barbara Hamlin

A View of Man in Measure for Measure as Seen Through The Imagery

McCrady, Bonnie Jones

The Elementary School Counselor as Perceived by Elementary School Principals in the Des Moines Independent Community School District

Padellford, Lynn D.

Elementary Curriculum Decision Making in Marion County, Iowa

Kramer, Duane Paul

The Professional Status of Boys Basketball Coaches in Iowa Class AA High Schools, 1968

Lipovac, Stephen A.

A Study of Principal Roles as Perceived by Elementary Teachers in The Des Moines Public Schools

Giese, David Berlen

EXPERIMENTAL TRICHINIASIS OF YOUNG
 
WHITE mICE BY STOMACH INTUBATION.
 
A Thesis
 
Presented to
 
The School of Graduate Studies
 
Drake University
 
In Partial Fulfillment
 
of the Requirements for the Degree
 
~~ster of Arts in Biology
 
by 
David Berlen Giese
 
August 1969
 
/0/
/ / $::7 
EXPERIl'iJENTAL TRICHINIASIS OF YOUNG
 
WHITE MICE BY STOMACH IN'I'UBATION
 
by 
David Berlen Giese 
Approved by Committee: 
studies 
.~ ) \f ) 
Ao" 
TABLE OF CONTENTS
 
PAGE
 
INTRODUCTION AND BEVIEW OF THE LITERATURE • • • • • •• 1
 
MATERIALS AND METHODS J
0 • • • • • • • • • • • • • • •• 
DISCUSSION • • • • • • • • • • • • • • • • • • • • • •• 6
 
CONCLUSIONS • • • • • • • • • • c • • • • • • • • • • • 11
 
LITEBATUBE CITED • • • • • • • • • • • • • • • • • • • • 13
 
LIST OF TABLES 
TABLE PAGE 
1. Intensity of Trichinizat10n of various age 
groups of young white mice. 8 
2. The determination of Chi-Square test as an 
analysis of experimental Trichinization 
data. 10 
LIST OF FIGURES 
FIGUBE 
PAGE 
1.	 Intensity of Trichinization of various age
 
groups of young white mice.
 9 
INTRODU eTION AND REVIEW OF THE LITERATURE 
The clinical history of Trichiniasis has been thor­
oughly described by Belding (1942). Leuchart and Virchow 
were the first to make significant progress in unraveling 
specific questions about the heLminth Trichinella spiralis. 
From the time they described the life cycle of this organism 
in the 1850's, as pointed out by Larsh (1963), it has 
remained one of the most intensively studied forms of life. 
Pertinent work has been done in the area of host 
resistance. Riedel (1948) demonstrated that there seemed to 
be a natural resistance that increased with age. His work 
showed that mice that were from 34 to 42 days of age when 
infected, yielded more than twice as many muscle larvae as 
those infected at from 5 to 6 months of age. All age groups 
yielded nearly the same number of adult worms, the difference 
being in the number of larvae produced and their invasion of 
host muscle tissue. 
¥mch of the current research is involved with 
resistance to repeated infections. Bass and Olson (1965), 
Ewert and Olson (1960), Mills and Kent (1965), Olson and 
Ewert (1961) and Olson and Hill (1966), have shown that it is 
difficult to demonstrate any great immunological reaction by 
the host toward T. spiralls. Most of these workers have 
dealt with crude extracts of adult worms and/or Excretion 
and Secretion antigens (ES antigens) in different age groups 
of mice. 
2 
Even though Silverman (1965) worked with rats, his 
findings are of interest. He found that !. spiralis larvae 
will mature and infect both the mother and her embryos if 
they are injected in. utero during pregnancy. He also found 
that if the larvae are injected into the amniott0 sac of each 
embryo, only the embryo is infected. 
While Bass and Olson (1965), Eerntzen (1965), Katz 
(1960), Khan (1966) and Larsh (1963) do not agree on the 
number of stages or on the time spent in each stage, they all 
have contributed greatly to the u~derstanding of the life 
cycle of T. spiralis. The phases of this organism's life 
cycle that are of particular importance to this study are the 
reproductive, the muscle invasion by the larvae and the 
subsequent encapsulation. Most of these researchers agree 
that the freed larvae will mature in the gut of their hosts 
from within 1 to 2 days. Stefanski and Przyjalkowski (1965) 
demonstrated that some bacteria in the intestinal flora seem 
to inhibit ecdysis and adult development, while others seem 
to favor their maturation. There is also an indication that 
location and retention within the gut may have an influence 
on resistance and acceptance of the infective larvae as cited 
by Campbell (1967). New muscle larvae can be found as early 
as the sixth day post infection, as pointed out by Khan (1966). 
Encapsulation may occur by the seventeenth day post infection 
as reported by Larsh (1963), but reaches its peak by the 
nineteenth or twentieth day. 
.3 
In terms of worm burdens, Bass and Olson (1965) deter­
mined that newborn mice seem to be more resistant to this 
infection than are adults. Silverman (1965) has shown that 
rat embryos in utero will accept challenging infections of 
Trichinella spiralis. Of the many questions still unanswered, 
one of particular interest here deals with host resistance. 
This study is an attempt to determine if there is a significant 
differenoe in the acoeptanoe of infections of T. spiralis in 
differing age groups of young white mice. 
MATERIALS AND METHODS 
Eight adult female and four adult male white mice were 
obtained from the Small Animal SUpply Company in Omaha, 
Nebraska. All of the mice arrived in Mequon, Wisconsin, 
August 7, 1967. 
Cages were made out of styrene plastic utility boxes, 
measuring 12 1/2" x 10 1/2" x 6 3/4". Each cage w·as divided 
into two equal halves by cementing a piece of the same type 
of plastic through the middle of each box. The covers were 
made out of 1/4" X 1/411 hardware cloth. Food hoppers 'tITera 
placed in each lid so that the Purina Mouse Chow was avail­
able ~ libitum. Water bottles were also placed in each cage 
so that water was available at all times. All mice were kept 
in a basement maintained at a temperature of apprOXimately 
740 Fahrenheit. 
4 
The stock female mice were bred and were separated 
from their oage mates shortly before delivery. The young 
were kept with their mothers until weaned, at which time the 
mothers were put back with the other adult females to be used 
again as breeding stock. Eaoh of the young in a litter was 
numbered by cutting a particular toe off of a given paw. 
This was done so that each individual in every litter could 
be identified from the time of infection tp the time of sacri­
fice. 
The larvae were maintained in some stock mice; however, 
the carcasses of experimental mice were used more often. An 
acidified pepsin digest solution was made up by adding 3 gm 
pepsin, N.F. powder (~~theson, Coleman and Bell) and 2 ml of 
conoentrated Hel to distilled water to make up a total volume 
of 200 ml. The viscerated carcasses of infected mice were 
ground in a Model 477 Series B Osterizer Blender for 20 
seconds at a speed indicated as number 2. The carcasses 
were suspended 1n part of the digest fluid while being ground. 
This slurry was then maintained at a temperature of 370 
Centigrade for from three to four hours in a Blue M "BUdget" 
bacteriological incubator. The fluid was then filtered 
through several layers of sterile gauze to remove bone and 
other material fragments from the larvae. The dilution 
method employed by Riedel (1948) was used to separate other 
materials by allowing the worms to settle to the bottom upon 
standing. The supernatant was removed by decantation and the 
5 
remaining portion was diluted using warm tap water until the 
fluid became relatively clear. The larvae were counted on a 
pyrex spot plate and appropriate larvae added or removed so 
that there remained fifty larvae per depression. The volume 
of fluid containing the larvae varied from 0.1 ml to 0.01 ml 
according to the age of the recipients, the younger mice 
receiving the smaller volume. Larva that had been isolated 
for more than eighteen hours were not used because Mills 
and Kent (1965) reported they lost infectivity during incu­
bation. 
Since oral intubation worked well for Bass and Olson 
(1965), it was chosen for use in this study. A minimum of 
twenty experimental individuals in each of five age groups. 
varying in increments of five days, were infected by oral 
intubation of 40-50 larvae. A tuberculin syringe with a 
blunted 20 gauge needle was used to deposit the larvae in 
the stomachs of the twenty. fifteen and ten day old experi­
mental groups. Because of their limited size. the five and 
one day old mice were intubated by using a drawn glass pipet. 
Within each litter one mouse was used as a Viability control 
and another as a placebo control. The placebo control 
received the same volume of tap water as the experimentals, 
but void of larvae. 
All experimental and control animals were kept for a 
minimum of thirty days post infection so that cyst walls 
might be well formed. Each individual was sacrificed by a 
6 
sharp blow to the back of the head and necropsied by the 
standard diaphragm press, similar to the procedure used by 
Riedel (1948). A Bioscope (Model N~ 60) with a 5X lene was 
used to check each diaphragm for the approximate number of 
larval cysts. This number was recorded and the diaphragm 
was then placed with the others of the same age group. A 
20 per cent formalin solution was used as a preservative. 
DISCUSSION 
It was found that not all larvae. in either the 
hypodermic needle or the glass pipet. were expelled when 
introducing them to the recipient hosts. The number 
remaining in the needle or pipet could be determined by 
flushing it and checking the wash water for larvae. From 
observation of this procedure it can be said that anywhere 
from 40 to 50 larvae were transferred to each host •. 
While fresh the diaphragms of all experimental and 
control animals were checked microscopically and pressed 
between two microscope slides. The total number of cysts 
were counted and rated as to a light, medium or heavy 
infection. A light infection was designated as having from 
1 to 25 cysts. medium 26 to 50 cysts. and a heavy infection 
having 51 or more cysts per diaphragm. It should be noted 
that all control animals were negative for the presence of 
any cysts. The results of the cyst counts were recorded and 
tabulated in Table 1. It summarizes the comparison of the 
7 
intensity of infection among the various age groups of mice. 
It shows both the total number and percentages of diaphragms 
in each category. As an example, there were 27 experimental 
hosts in the 1 day old group. All proved to be positive, but 
not to the same degree. As can be seen ~4 were heavily 
infected, 5 medium and 8 lightly infected. These numbers 
expressed as a per cent of the total 27 are: 52%, 18% and 
30% respectively. It can also be seen, by following each 
category across the table, that there were 52% heavy in the 
1 day, 74% in the 5 day, 25% in the 10 day, 73% 1n the 15 day 
and 62% heavy infections in the 20 day group. Each category 
can be analyzed for the number and percentage light, medium 
or heavy infections by this manner. The per cent in each 
category can also be seen graphically in Figure 1. It 
illustrates each of the three categories, (heavy, medium, 
light), as well as the total per cent positive in each group. 
It is interesting to see that in all groups except the 10 day 
the heavy infection category has the greatest percentage. It 
can be seen that the light infection category seems to 
decrease with age. The per cent positive shows little to no 
signlfic8~t variance from group to group. 
Tne data was also analyzed by the use of the Chi­
Square test, as seen in Table 2. The one and five day old 
groups could not be analyzed by this method because they 
were all positive, thus they had no deviation. The ten, 
fifteen and twenty day old groups, showed Chi-Square values 
TABLE 1. Intensity of Trichinization of various age groups of 
young white mice 
Total number observed 
1 DAY 
f<1 
OLD 5 DAY OLD *10 DAY OLD 15 DAY OLD **20 DAY OLD 
No. % No. % No. % No. % No. % 
27 100 23 100 24 100 33 100 29 100 
Total number positive 27 100 23 100 21 88 29 88 26 90 
Total number negative 0 0 a a 3 12 4 12 3 10 
HEAVY Trichinization 
(51 or more cysts per 
d,iaphragm) 
14 52 17 7~ 6 25 24 73 18 62 
i~DIUM Trichinization 
(26-50 cysts per 
diaphragm) 
5 18 4 17 14 58 5 15 8 28 
LIGHT Trichinization 
(1-25 cysts per 
diaphragm) 
8 30 2 9 1 4 0 0 0 0 
1~One mouse died the day of infection, thus it was not included 1n the data analysis. 
**One mouse died on the third day post infection, thus it was not inclUded in the data. 
analysis. 
co 
ft.">i,;" «, ~,t'I 1!i--'I7' l!'f"0"-:!" ~_ 1'!< r,-t· "ti,'! 'l!' ''1'1: ~ '& 
9 
1 DAY 5 DAY 10 DAY 15 DAYOLD OLD OLD OLD 
100 
l-
5:l 
0 90 .oM 
.p 
:as 80N . 
oM 
s::: 
oM 70 
..t:: 
0 II
I
oM 60 I 
~
 
I
 
~
 I 
I 
I I
 
I ~
 I .
I 
•
II
 -I ~ 
20 DAY 
OLD 
, 
..I-l I8 • I
~ 50 ·i0 I'I I 
(1) 40 ~ to .~ .~aJ
 
.p 30
 ~ .~s::: .~ 
(]) I§0 20 
I 
~H ~ •(]) .~ 
t:Lt 10
0 
.~~ ·1 
~ 
II~I
 I.:...!~ 
Figure 1. Intensity of Trichinization of various age 
groups of young white mice. 
KEY. ~~~ Per cent HEAVY Trichinization (51 or more cysts per 
diaphragm) • 
~ Per cent ~lliDIUM Trichinization (26-50 cysts per 
diaphragm) • 
_.- Per cent LIGHT Triohinization (1-25 cysts per 
d iaphragm) • 
~ Total per cent positive. 
10 
TABLE 2. The determination of Chi-Square test as an 
analysis of experimental Trichinization data 
Experimental Groups 
1 DAY OLD 
5 DAY OLD 
10 DAY OLD 
15 DAY OLD 
20 DAY OLD 
Deviation Squared (X - m)2 
0 
0 
9 
16 
9 
(X - m)2 
m 
0.0 
0.0 
0.376 
0.486 
0.310 
11 
of 0.376, 0.486 and 0.310 respectively, as seen in the last 
column of Table 2. These va1ues can be expressed as prob­
ability factors (p factors) of 0.6, 0.5 and 0.7. This means 
that the results of these three groups could be expected (on 
the whole) to be the same from 50 to 70 per cent of the time. 
CONCLUSIONS 
This study attempted to determine if there is a signi­
ficant difference in the acceptance of infections of Trichin­
~ spiralis in differing age groups of young white mice •. 
The ages of the mice varied in five day increments so that 
some were one day old, five days old, ten days old, fifteen 
days old and twenty days old when infection was attempted. 
It has been clearly shown in this investigation that 
there was no significant differenoe in the acceptance of 
infections of T. spiralis. It is likely that errors in 
technique contributed to the 10-12 per cent deviation from 
the 100 per cent level in the experimental mice ten days and 
older. 
As a result of the diaphragms examined, it is recom­
mended that a more precise technique be used in determining 
the number of larvae introduced and the subsequent invasion 
of host tissues. In this study only the diaphragm was 
examined. Katz (1960) however mentioned in his study that 
when a diaphragm press proved negative, he did a total digest 
and examined the fluids for the presence of larvae. SOIDe of 
12 
those that seemed negative in this study may have shown that 
while the diaphragm was negative there was infective larvae 
elsewhere in the caroass of the host. It would be of value 
to use the total digest technique in order to obtain more 
precise data in terms of the total number of larvae accepted 
by each host. Other suggestions are to increase the number 
and age range of hosts sampled and to vary the number of 
larvae introduced into each host. 
LITERATURE CITED
 
Bass, G. K., and L. J. Olsen. 1965. Trichinella spiralis in 
newborn mice; course of infection and effect on resist ­
ance to challenge. J. Parasito!. 51:640-644. 
Belding, David. 1952. Textbook of clinical parasitology. 
Appleton, Century, Crofts, Inc., New York. 
Berntzen, Allen K. 1965. Trichinella spiralis in vitro and 
in vivo, with a redescription of the life cycle. Exp. 
Parasitol. 16:74-107. 
Campbell, William C. 1967. Distribution of Trichinella 
spiralis in the small intestine of young mice. J. 
Parasitol. 53:395-397. 
Ewert, Adam, and Leroy J. Olson. 1960. Immunological toler­
ance stUdies with mice and Trichinella. J. Parasitol. 
46:849-854. 
Katz, Frank F. 1960. The oral transplantation of intestinal 
stages of Trichinella spiralis. J. Parasitol. 46:500-504. 
Khan, Zafer All. 1966. The postembryonic development of 
Trichinella spiralis with special reference to ecdysis. 
J. Parasitol. 52:248-259. 
Larsh, John E. Jr. 1963. Experimental triciniasis. Advance. 
Parasitol. 1:213-286. 
Mills,	 Charles K' f and Nairn H. Kent. 1965. Excretions and 
secretions of Trichinella s;piralis and their role 1n 
immunity. Exp. Parasitol. 16:300-310. 
14 
•
 
Olson. Leroy J., and Adam Ewert. 1961. Further studies on 
immunological tolerance with mice and Trichinella. Texas 
Rep. BioI. Med. 19:866-868. 
Olson. Leroy J. t and M. Hill. 1966. Resistance of mice 
injected at birth with extract of adult Trichinella 
spiralis to subsequent infection. J. Parasitol. 52:821. 
Riedel, B. B. 1948. Age resistance of mice to the nematode 
Trichinella spiralis. Amer. Microscop. Soc. Trans. 
67:268-271. 
Silverman, Paul H. 1965. In vitro cultivation procedures 
for parasitic helminths. Advance. Parasitol. ):179-181. 
Stefanski, Witold, and Zdzislaw Przyjalkowski. 1965. Effect 
of alimentary tract microorganisms on the development of 
Trichinella spirali~ in mice. Part I. Exp. Parasitol. 
16:167-173. 
Stefanski, Wltold. and Zdzislaw Przyjalkowski. 1966. Effect 
of alimentary tract microorganisms on the development of 
Trichinella BEiralis in mice. Part II. Exp. Parasitol. 
18:92-98. 


Experimental Trichiniasis of Young White Mice by Stomach Intubation

Burnett, Charles M.

Physical Education of The Educable Mentally Retarded for Cass County, Iowa

Hendricks, Judy Christine Aksland

Isolation and Characterization of Rat Liver Nuclear Envelopes

Hindley, Harvey Joseph

An Investigation into Student Pressures and Mental Health in Selected Elementary Schools of Story County

Poehlein, Barbara C.

The Effects of Student Teaching on The Educational Attitudes of Prospective Teachers

Drake University

Not a member yet