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THESIS
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ABSTRAC'

MIGRATIONS OF NEMATOSPIROIDES
DUBIUS (NEMATODA5 IN THE MOUSE

by James R. Ford

The migratory abilities of the infective larvae of
Nematospiroides dubius were studied by introducing the
larvae into the mouse host at the following sites; the
surface of the skin, the systemic venous circulation and
the peritoneal cavity. Oral infections were also studied
to determine if larvae migrate from the intestine. Larvae
were recovered from the organs and tissues by pepsin diges-
tion.

Only a few of the infective larvae were able to

penetrate the skin and complete the migration from the
subcutaneous layers of the skin to the intestine. When

infective larvae of E. dubius were injected into the sub-
cutaneous layers of the skin most of the larvae did not
enter the vascular system and only a few were found in the
small intestine, but the larvae did undergo random migration
in the tissues. Larvae injected into the tail vein were
trapped in the lungs and migrated from the lungs to the
digestive tract. Larvae of E. dubius possessed the same
ability as the skin penetrating larvae of Nippostrongylus

braziliensis to migrate from the lungs to the digestive

 

tract but fail to enter the vascular system from the tissues.

MIGRATIONS OF NEMATOSPIROIDES
DUBIUS (NEMATODA) IN THE MOUSE

By

James R. Ford

A THESIS

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

MASTER OF SCIENCE

Department of Microbiology and Public Health

1967

ACKNOWLEDGEMENTS

The author expresses deep gratitude to Dr. D. W.
Twohy for his assistance and guidance throughout this
study.

ii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . .
METHODS AND MATERIALS . . . . . . .
RESULTS . . . . . . . . . . . . . .
I. Oral Infection . . . . . .

II. Intravenous Injection.
III. Intraperitoneal Injections
IV. Subcutaneous Injections . .
V. Skin Penetration . . . . .
VI. Comparison of All Routes .
DISCUSSION . . . . . . . . . . . . .
SUWARY o o o o o o o o O o o o o 0
CONCLUSION O O O O O O O O O O O O 0

LITERATURE CITED . . . . . . . . . .

iii

Page

l2
l2
15
18
19
21
26
31
32

33

LIST OF TABLES

Table Page
1. Recovery of adult N. dubius from the small
intestine after infectIon By various routes.

The percentages are averages of groups of
five mice each . . . . . . . . . . . . . . . . 25

iv

Figure

1.

LIST OF FIGURES
Page

Location and numbers of worms recovered at

intervals of time after oral intubation of
exsheathed infective larvae of N. dubius.

Each point represents an average from 5 mice. . 14

Location and numbers of worms recovered at

intervals of time after intravenous injection

of infective larvae of N. dubius. Each point
represents an average from 5 mice. . . . . . . 17

Location and numbers of worms recovered at

intervals of time after subcutaneous injection

of exsheathed infective larvae of N. dubius.

Each point represents an average from 5 mice. . 23

INTRODUCTION

Nematospiroides dubius is an intestinal nematode of
mice which has come to the attention of several investiga-
tors in recent years because of the pathology and immunolog-
ical response caused by this worm. Lepak (1962) suggested
that some of the infective larvae of'fi. dubius introduced
into the subcutaneous layers of mouse skin were able to
migrate to the intestine and mature into adult worms. This
indicated that‘fl. dubius may have some of the tissue penetra-
ting abilities of the skin penetrating nematode £1222:
strongylus braziliensis.

The purpose of this investigation was to determine
what abilities for tissue penetration and migration.y.
dubius shares with N. braziliensis. To do this infective
larvae of N. dubius were injected into various tissue sites
and the locations of the larvae followed to determine the

extent and route of their migration.

LITERATURE REVIEW

Nematospiroides dubius was originally described by

 

Baylis (1926) from the small intestine of the wood mouse
(Apodemus_§ylvaticus) and placed in the family Heligmosomatidae.

 

‘N. dubius has since been reported from other wild mice like
Peromyscus maniculata (Ehrenford, 1954) and Mus musculus
(Spurlock, 1943) and can be easily maintained in several
strains of laboratory mice.

The life cycle of N.‘gg§ig§ as described by Spurlock
(1943), Ehrenford (1954), Baker (1954), and Callizo (1962)
takes about 15 days under optimal conditions. Fahmy (1956)
investigated the free-living stages of N. dubius and found
that the eggs hatch within 24 hours after being passed with
the feces. Third stage larvae were found 48 to 56 hours
later, but they were not infective until at least 5 days
after the eggs hatched. Fahmy stated that mice became
infected by ingesting the sheathed third stage larvae.

Liu (1965a) found larvae in the mucosa and submucosa of the
stomach until 56 hours after ingestion. Exsheathed larvae
were found by Baker (1954) and Liu (1965a) in the crypts
of Lieberkhhn of the small intestine 24 hours after ingestion.
The larvae first penetrated into the mucosa through the crypts
causing necrosis at the sites of entry. This was followed by
migration into the muscularis where, according to Ehrenford
(1954), they came to rest against the longitudinal muscle

2

3
layer. Baker (1954) believed that the transverse orienta-

tion of the larvae to the longitudinal axis of the small
intestine indicated that the larvae encyst in the circular.
muscle layer. Liu (1965a) reported that by the second day

of infection petechial and ecchymotic hemorrhages were seen
in the muscularis and worms were found at the sites of
hemorrhage. Baker (1954) stated that the size and number of
sites of hemorrhage increased up to the third day of infec-
tion because of the rapid growth of the larvae in the
muscularis and the entry of more larvae into the tissues.
Leucocytic infiltration began causing the sites of hemorrhage
to become opaque. Progressive tissue degeneration and
leucocytic infiltration was followed by encapsulation of the
larvae in the muscularis during the second and third day of
infection. The sites of encapsulation increased in size
until the twelfth day due to the continued infiltration of
leucocytes. During the period between 3 to 12 days after
infection, the gut wall was greatly distended. Spurlock
(1943) reported that some animals died because of perforation
of the gut wall. Deaths also occurred from mass hemorrhage
of the mucosa and fecal occlusion of the intestine of heavily
infected mice. The mass hemorrhage of the mucosa occurred
when the larvae returned to the lumen of the small intestine,
6 to 8 days after infection. The adults attached to the
mucosa with the greatest number near the pyloric valve.

Fifteen to 16 days after infection unembryonated eggs were

4

passed in the feces of the mice.

Lesions develOped in other organs besides the intestine
3 to 9 days after infection. Spurlock (1943) reported that
the liver, kidneys, and spleen were pale in color and that
the spleen was enlarged. There was "a slight serous to
serosanguinous exudate in the body cavity". Baker (1963)
and Liu (1965) both reported that leucocytosis and
spleenomegaly occurred early in the infection due to "necrotic
substances" produced by the larvae in the intestinal wall.
Liu reported that the mesenteric lymph glands were heavily
infiltrated and hyperplastic. The endothelial cells in the
branches of the portal viens were swollen and neutrophils
were evident around these vessels. Other neutrophilic foci,
which were observed in the parenchyma of the liver, increased
in numbers until the ninth day. Twelve to 21 days after
infection these lesions became progressively fibrotic; first
those in the capsule of the liver followed by those near
the branches of the portal vien sinusoids. Liu failed to
find larvae in sections of the mesenteric lymph glands,
liver, or spleen showing pathology.

Only a few experiments to determine the migration
pattern of the infective larvae introduced at abdominal
sites in the host have been published. Roman (1951) examined
histological sections of skin of rats exposed to larvae.
Since he did not find larvae he concluded the larvae would
not penetrate the skin. Baker (1955) injected third stage

larvae both into subcutaneous areas of the abdomen skin and

5

into the peritoneal cavity. Some of the larvae invaded and
encysted in the mesenteries of the mice and showed signs of
growth, but no evidence of sexual develOpment was observed.
He also noted subcapsular lesions lacking larvae in the
liver and Spleen and stated they were due to larval penetra-
tion of the capsule. Lepak et. al. (1962) made incisions
in the flanks of mice which eXposed the subcutaneous areas
of the skin. Larvae were introduced into these incisions
and the skin was closed with sutures. When some of the mice
were autOpsied l4 and 28 days later, worms were found in the
subcutaneous tissues and the gut. Stunted worms were found
in the subcutaneous tissues 185 days after initial infection.
The males had well develOped bursae and some of the females
contained infertile eggs. Lepak (1962) wrote, "Further
experiments will be necessary to show whether the few worms
found in the gut were the result of migration" from the skin.
The life cycles of only a few members of the Family
Heligmosomatidae are known, but two routes of larval entry
into the host have been demonstrated: skin penetration and
ingestion (Skrjabin et. a1. 1952). Yokagawa (1922) and
Schwartz and Alicata (1945) reported the Nippostrongzlus
braziliensis, which normally infects its host by skin
penetration, can infect rats by the oral route. Longistriata
musculi which normally enters the mouse by ingestion has also
been reported by Schwartz and Alicata (1955) to infect by
skin penetration. Unfortunately their observation have not

been repeated by other investigators. The recovery of

6
'N. dubius adults from the small intestine after the larvae
were placed in the subcutaneous tissues indicates that both
routes of infection might be possible for this species
(Lepak, 1962).

For infection by either the oral or cutaneous route a
nematode must have certain adaptations or abilities; such as
the ability to select the successful route of migration and
to penetrate tissues. To be a successful skin penetrator
like N. braziliensis the infective larvae must be able to
exsheath in the external environment or in the absense of
stimulatory conditions peculiar to the digestive tract and
penetrate the host's skin. Enzymatic action may be required
for penetration as has been demonstrated with Strongyloides,‘
but the infective larvae of‘fl.‘bg§§iliensis apparently lack
most common enzymes associated with penetration (Lewert 1954).
Once the larvae has entered the host it must be able to follow
a migratory path to the site of final maturation, which is
the lumen of the gut in most cases. The larvae of
N. braziliensis, like most skin penetrating forms, must
enter the vascular system to be carried to the lungs. They
must be retained in the capillary bed of the lungs or else
they would be carried to all parts of the body by the
‘ systemic circulation. Retention in the lungs may be due
to their inability to pass through the capillaries. The
larvae in the lungs gain access to the air passages, either

by active migration or by rupture of blocked capillaries,

7
where they migrate or are carried by ciliary action up the

trachea and are eventually swallowed. Once in the gut the
larvae actively enter or adhere to the mucosa of the small
intestine.

There would seem to be less adaptation necessary for
oral infection. Forms ingested orally like N. dubius are
stimulated to exsheath by the conditions of the physio-
chemical environment encountered in the host. Extensive
tissue migrations are not common after oral infections.
Some species in the Trichostrongyloidea like‘fl. dubius may
penetrate into the mucosa and enter the muscle layers and

eventually return to the lumen.

METHODS AND MATERIALS

The parental stock of Nematospiroides dubius was
obtained from the Ely Lilly Company and has been maintained
in this laboratory for about 5 years. All mice used for
experimental purposes were female albino mice of the Swiss
Webster strain weighing between 15 and 20g. Larvae were
obtained by smearing moist fecal pellets containing eggs on
strips of filter paper and culturing them in centrifuge tubes
as described by Sadig (1964). The infective, 5rd stage
larvae were collected from 6 day old cultures, suspended in
0.85% NaCl and washed free of most of the fecal debris by at
least 4 low Speed centrifugations and decantations. After
the final wash the larvae were suspended in 5 to 7 ml of 0.85%
NaCl and stored at 4°C in 50 ml Erlenmeyer flasks stOppered
with cotton plugs.

Stock infections and some experimental infections were
given by oral intubation of infective larvae using a two inch,
blunt, curved 18 gauge needle attached to a 1 ml glass
tuberculin syringe. For stock infections mice were given 200
to 500 larvae each and the infection was transferred at 5
month intervals.

Except for certain oral infections or when otherwise
specified, all larvae used for experimental infections were
exsheathed. Nearly one hundred percent exsheathment of the
larvae was obtained by first suspending the larvae in a
0.154 M KCl solution adjusted to a pH of 1.7 with 0.154 M HCl.

8

9

One ml of the larval suspension was then placed in a 10 x 15
mm test tube. Rubber serum bottle stoppers were used to

seal the tubes. Two hypodermic needles were inserted into

the tube, one as an inlet and the second as an outlet for gas.

A mixture of 5% C0 and 95% N2 was bubbled into the larval

2
suSpension for ten minutes before the needles were removed.
The larvae were then incubated at 59°C for 40 minutes while
exsheathment took place. After exsheathment the larvae were
washed 4 times by a series of low Speed centrifugations and
decantations. The best exsheathment occurred after the larvae
had been stored for at least 7 days at 4°C.

The larval concentration in the suspensions used for
infection of mice was determined by withdrawing at least
four 0.05 ml or 0.1 ml samples with a 1 ml tuberculin syringe,
counting the larvae in a Scott Chamber, and averaging the counts.
The larval suspension was adjusted to give the desired number
of larvae in 1 ml of suspension.

Experimental oral infections were given to the mice in
the same manner used in stock infections. The abdomen of
each mouse was rubbed thoroughly with 70% ethyl alcohol before
and after each subcutaneous and intraperitoneal injection of
larvae to kill any larvae that might have been accidentally
deposited on the skin. For intravenous injections mice were
restrained in a box with a U-shaped notch to allow the tail
to protrude. The injections were given in the right lateral
tail vein using a 26 guage needle. The tail was wiped before
and after each injection with 70% ethyl alcohol. To test the

10
ability of the larvae to penetrate the skin, the mice were
anesthetized with 0.1 cc of a 1:100 dilution of Halatal (1
grain Na pentobarbital/cc carrier) by intraperitoneal injections.
The abdomen of each mouse was shaved with electric clippers
fitted with a size 40 head. Animals which received cuts or
abrasion from shaving were not used. After shaving the mice
were tied to a table with thread and tape and the eXposed abdomen
was wiped with 70% ethyl alcohol. When the alcohol was dry the
larvae were spread over the shaved area. The mice were exposed
to the exsheathed infective larvae for 45 to 60 minutes before
the abdomen and the surrounding fur was thoroughly wetted with
70% alcohol. After 5 minutes exposure to the alcohol the
mice were placed back in their cages. The mice were kept at
59° from the time they were anesthetized until they were
totally revived to prevent a drop in body temperature.

To determine the fate of the larvae introduced at the
various sites the lungs, trachea, eSOphagus, stomach, small
intestine, liver, and kidneys were checked for larvae. The
skin at the abdominal site of infection was checked in those
eXperiments involving skin penetration, subcutaneous injec-
tions and peritoneal injections. The subcutaneous tissues
and the abdominal muscle layers were included in each of the
skin samples examined.

The larvae were recovered after pepsin digestion of the
tissues. The organs and tissues were placed in separate

114 x 25 mm (40 m1) glass screw cap vials and chopped into

11
small pieces with a pair of scissors. A pepsin solution
(1% pepsin, 0.5% HCl, and 0.85% NaCl) was added until the
fluid filled all but the upper 1 cm of the vial. The vials
were capped and agitated in a shaker box at 59°C in a room
incubator for 6 hours. After digestion the contents of the
vials were placed in 50 ml centrifuge tubes and centrifuged
at 72 X G for 5 minutes. The supernatant was carefully
decanted and the settled contents were resuspended with 0.85%
NaCl and centrifuged again. This was repeated until the
supernatant was relatively clear. All except the bottom
15-20 ml of supernatant was then decanted from the centrifuge
tube. The remaining volume was stirred and poured into a
petri dish marked off in % inch squares. In the experiments
that large numbers of larvae were recovered only 20% of the dish
was examined. When larvae were few, the entire dish was
examined. All counts were made using an A.0. Cycloptic

dissecting microscope.

RESULTS

Oral Infection

Mice were infected orally with sheathed infective
larvae of N. dubius in order to confirm prior descriptions
of larval migration in the host, to determine the
efficiency of the techniques employed in recovering worms,
and to search for larvae in other body organs that might
indicate aberrant migrations. Eighteen mice were infected
orally with 185 larvae each and autopsied in groups of 5
at l, 2, 5, 4, 6, and 10 days after infection. During
the first 5 days less than 20% of the larvae were recovered
from the stomach and small intestine (Fig. l). Digests
of the stomachs revealed the greatest number of larvae
at 24 hours. Only a few larvae were found in this organ
on the second and third days after infection. Larvae
were in the intestine as early as 24 hours after infec-
tion, but the recoveries of larvae were low and variable
during the first 5 days of the infection. After 5 days
the percent recovered increased with the age of the
infection. Ten days after infection 94% of the larvae
were recovered as adults from the small intestine. The
lungs, trachea, esophagus, liver and kidneys of all
animals employed in this experiment were removed, digested,
and examined to determine if larvae migrated from the
small intestine. All examinations for larvae were negative.
The lungs, liver, and kidneys were selected because of

12

Figure l.

13

Location and numbers of worms recovered

at intervals of time after oral intubation
of exsheathed infective larvae of

.E' dubius. Each point represents an

average from 5 mice.

 

NO. OF wows RECOVERED

20° SMALL mresrme .- ——
STOMAOH . -----

I75 INFECTION l85 LARVAE

I5 0
e
l25
l o o
O
75
50
25 V\.
’I’ . \\‘°. ~
I 2 3 5 6 7 8 9 IO

TIME IN DAYS

II.

15
their large blood flow and extensive capillary beds
which would be expected to filter out larvae entering
the vascular system.

It was apparent from the results of this experiment
that many of the young larvae were lost due to their
small size or to the action of the pepsin solution on
the larvae.

Intravenous Injection

Exsheathed infective larvae of N. dubius were
injected intravenously into mice to determine if they
were capable of a lung to digestive tract migration.
Large numbers of larvae were found in the lungs 5 hours
after intervenous injection (Fig. 2) and the number of
larvae remained high for 2 days. From 5 days on the
lung recoveries decreased, but some larvae persisted and
a few even showed development into late fourth or early
fifth stage larvae. Larvae were first recovereed frOm
the trachea, esophagus, and stomach 24 hours after
infection. Small numbers of larvae were found in‘these
organs up to 6 days after infection. There was no marked
increase in the number of larvae in these sites 2 to 5
days after infection when the majority of larvae
disappeared from the lungs. The worms were first seen
in the small intestine 2 days after infection. After
2 days the number of worms recovered from the small
intestine rose gradually at first with a dramatic increase

in recoveries after 6 days, the time larvae migrate out

16

Figure 2. Location and numbers of worms recovered
at intervals of time after intravenous
injection of infective larvae of‘N. dubius.
Each point represents an average of 5

mice.

 

no. or weeus Recovseeo

I75

I50

I25

IO

75

25

 

LUNGS =
ESOPHAGUS

8 TRAOHEA 8 -----
STOMAOH 3 ""—

INTESTINE ' —'—
INFECTION 500 LARVAE

/ /'

/\ "/,.:>~<:// /

/ , .A

I 2 3 ' 5 6 7 8

1 '0‘----

TIME 'IN DAYS

III.

18
of the intestinal wall into the lumen. Checks of the
liver and kidneys failed to find any larvae, indicating
the larvae had not entered the systemic circulation.
Thirty-three percent of the larvae were found as adults
10 days after infection. The larval recoveries were
generally lower than those of the adults, with the
lowest number of larvae recovered between 5 and 6 days
after infection. The results show that at least some
of the exsheathed larvae are capable of a lung to diges-
tive tract migration and that most of the larvae given
intravenously have a period of residency in the lungs.

Intraperitoneal Injections

 

Larvae were injected into the peritoneal cavities
of mice to determine: (1) if they could migrate to the
small intestine to mature to adults and (2) if they
reached the intestine, what migratory route they would
follow to the intestinal wall. A group of 24 mice were
injected with 520 larvae each and sacrificed in groups
of 5 at predetermined times. Twenty-seven larvae were
recovered in peritoneal washings 5 hours after infections,
but no larvae were recovered from peritoneal washings
of mice killed at 6 hours or 1, 2, 5, 6, 10, or 18 days
after intraperitoneal injections. Examinations of the
abdominal tissues, lungs, trachea, esophagus, small
intestine, liver and kidneys after pepsin digestion
failed to demonstrate any larvae. Digestions of

the stomachs led to the recovery of 9 undeveloped

IV.

19
larvae from the mice autopsied at 5 days and 5 undeveloped
larvae from those examined 6 days after infection.

Subcutaneous Injection

 

The ability of larvae to migrate from the sub-
cutaneous tissues was checked by injecting larvae under
the abdominal skin and attempting to find them in pepsin
digests of the abdominal skin and muscle, lungs, trachea,
esophagus, stomach, small intestine, liver, and kidneys.
A group of 21 mice were given subcutaneous injections of
500 exsheathed larvae. They were sacrificed in groups
of 5 at 5, 5, 10, 15, 20, 50, and 45 days after infection.
Most of the recovered larvae were found in the sub-
cutaneous and muscle layers of the abdomen, however,
lateral migration did occur. During the ten day period
after infection mostly undeveloped larvae (third stage
to early fourth stage larvae) were recovered from the
subcutaneous tissues (Fig. 5). After this period the
number of undevelOped larvae decreased while the number
of developing larvae (late fourth stage to fifth stage)
increased. Many of the later worms were fully mature
as was demonstrated by the well developed bursae of the
males and the presence of eggs in the females and
surrounding tissues. Few larvae or adults were recovered
from the small intestine and no recoveries were made
from digests of the lungs, trachea, esophagus, stomach,
liver, or kidneys. In one mouse 2 larvae were found in

the mesenteries between the pancreas and the small

Figure 5.

20

Location and numbers of worms recovered
at intervals of time after subcutaneous
injection of exsheathed infective larvae.
Each point represents an average from

5 mice.

lThird to early fourth stage larvae.

2Late fourth stage larvae to fifth

stage worms.

 

NO. OF wows RECOVERED

200

I75

I25

25

SUBOUTANEOUS TISSUES

UNDEVELOPED' = ----
DEVELOPED‘ . __
SMALL INTES‘I’INE . ——

INFECTION 300 LARVAE

O
’ \
// \
/ / \
y \
I \
I, \\
9. °
/ x I
’1’ “ I
e’ “‘01,
I
; ,J-‘\
I, / \
I, / - . ---- -‘
IO 20 30 40

TIME IN DAYS

22

intestine. The highest recovery of adults from the
small intestine was 1% of the larvae injected. Forty—
five percent of these same larvae were recovered as
adults from 5 mice after oral infection. The route of
migration that was followed to reach the small intestine
after subcutaneous injection of larvae could not be
determined because of the small number of successful
migrants.
Skin Penetration

Exsheathed larvae of N. dubius were placed on the
shaved abdomens of mice to determine if they could
penetrate the skin. In one experiment 1000 larvae were
placed on the exposed abdomens of 8 mice. Three of these
mice was autopsied after 10 days and the abdominal
tissues, lungs trachea, es0phagus, stomach, small
intestine, liver, and kidneys were digested with pepsin
solution. Eighty-four (5%) of the larvae exposed to the
skin were recovered as adults in the small intestine and
6 larvae were found in the abdominal subcutaneous tissues.
Eggs were found in the feces of the remaining mice. In
an attempt to determine the route of migration to the
intestine, the abdomens of 24 mice were exposed to 500
exsheathed larvae each and then autopsied in groups of
5 mice each, 6, 9, 15, 15, l7, l9, and 26 days later.
Adult worms were seen in the small intestine at 15, 15,

19, and 26 days, but the adults present accounted for

VI.

23

less than 1% of the larvae exposed to the skin. No
worms were found in pepsin digests of the abdominal
tissues, lungs, trachea, esophagus, stomach, liver, or
kidneys. I
Comparison of All Routes

An attempt was made to determine if the variation
in recoveries after different routes of infection was
not due, in part, to differences between larval cultures.
Seven groups of 5 mice each were infected with one of
two larval preparations. All of the mice were autopsied
14 days after infection and the number of worms in the
small intestine counted. The route of infection, larva1_
culture employed, the presence or absense of sheaths,
and the adults recovered in each group are listed in
Table l. The percentage of adults recovered in the
groups receiving oral injections was similar. Two
groups received intravenous injections; one with
exsheathed larvae and a second with sheathed larvae.
The percentage of larvae maturing to adults after
intravenous injection was much lower than by the oral
route, but the recoveries were comparable for infections
with sheathed or exsheathed larvae. Mice exposed to
infection by skin penetration or by subcutaneous injection
both showed very few adult worms, but the percent
recoveries were of comparable magnitude (2% and 3%).

The final recoveries of adults in this eXperiment

were comparable to the results of earlier experiments.

24
It would seem that the variance in infectivity between

larval cultures was small.

25

TABLE 1. Recovery of adult N. dubius from the small
intestine after infection by various routes.
The percentages are averages of groups of
five mice each.

 

 

 

Route of Larval Inffiective 76 Adults

Infection Culture Larvae Recovered

Per 05 I 230 99%
exsheathed

Intravenous I 250 41%
exsheathed

Subcutaneous I 250 5%
exsheathed

Percutaneous I 250 2%
exsheathed

Per OS I 230 96%
sheathed

Intravenous II 220 59%
sheathed

Per OS II 220 94%

sheathed

 

 

DISCUSSION

As a method for recovering Nematospiroides dubius larvae
from host tissues, the technique of pepsin digestion had some
drawbacks. Only 10-25% of the third and fourth stage larvae
could be found, with the lowest recoveries occurring 5 days
after oral and subcutaneous infection and 5-6 days after
intravenous infection. Increased recoveries were associated
with the appearance of late fourth stage larvae. As the
larvae matured to adults the recoveries increased after both
oral and intravenous infection, exceeding 90% with oral
injections. This indicated that the larvae were present
but only a small percent were found. Third and early fourth
stage larvae could have been lost either in the process of
centrifugation and washing of digests or of digestion of
the tissues. Partial digestion could alter the Specific
gravity and contribute to a loss in the centrifugation
process if the early stages were more subject to digestion
than late stage larvae, When infective larvae of.N. dubius
were intubated orally the larvae followed the general course
of migration described by Spurlock (1945), Ehrenford (1954),
Baker (1954), and Callizo (1962). The percentage of larvae
that survived to form adults was high, above 90%, whether
sheathed or exsheathed larvae were employed. This is com-
parable to the 85% survival recorded by Baker (1955, 1954)
and above 90%»reported by Spurlock (1943). Since larvae
were not found in organs other than the digestive tract

26

27

after oral infection, it was concluded that N. dubius
has no tendancy to migrate from the digestive tract.
This would be consistent with a Trichostrongylidae-
type of life cycle rather than Nippostrongylus
braziliensis type.

N. braziliensis is the only other Heligmosomid whose
migratory route has been studied in any detail. Schwartz
and Alicata (1954) and Yokogawa (1922) reported success in

infecting rats with N. braziliensis by the oral route; but

 

lung migration preceded establishment in the small intestine,
and the percentage of infection was low. Simaren (1964)
compared the percentage of survival ole. braziliepsis larvae
after different routes of infection. He found that about
14% of the larvae survived after oral infection, whereas

69% survived after subcutaneous injections. The behavior of
,N. braziliensis after oral infection differs greatly from
that of N. dubius in that they still undergo a lung migration
before becoming established in the small intestine.

When the infective larvae of N, dubius were injected
into the tail vein, it appeared as though most of them were
carried to the heart and lungs via the inferior vena cava
and pulmonary artery. Since no larvae were found in the
liver and kidneys, all organs of large blood flow and
extentive capillary beds, it was presumed that nearly all
of the larvae were retained in the lungs. The larvae

migrated from the lungs into the trachea, esophagus, and

28
stomach in the greatest numbers from about 24 to 48 hours
after injection. Once the larvae arrived at the small
intestine the pattern of develOpment was similar to that
of oral infection, however, only about 40% of the larvae
could be recovered as adults. The reasons for the lower
percentage of adults could not be ascertained. The
recoveries of adults were about the same whether sheathed
or exsheathed larvae were employed. Either the sheaths did
not inhibit this migration or they exsheathed somewhere in
the process of migration. It is significant that N. dubius
larvae which are adapted for early develOpment in the
_intestine are also able to undergo a lung to digestive tract
migration. The time required for migration from the lungs
was comparable to that of N. braziliensis (Twohy, 1956), where
most of the larvae leave the lungs within 50 hours after the
subcutaneous injection of larvae. Simaren (1964) found that
57% of the infective larvae of N. braziliensis injected into
the tail veins of rats survived to adults. This is compar-
able to the 41% survival found for N. dubius by the same
route in this investigation. The infective larvae of
N. dubius appear similar to those Nebraziliensis in their
ability to migrate from the lungs. It could not be determined
if the lung-digestive tract migration was due to a passive
response in which the capillaries rupture, the larvae enter
the reSpiratory tract and are carried upward by the ciliary
current or to a basic behavior pattern of both N.

braziliensis and N. dubius.

29
The results of larval survival in the skin after
subcutaneous injection confirms Lepak's (1962) observations,
except that more lateral migration was noted and some worms
were found 1 inch or farther away from the site of injection.

The infective larvae of N. braziliensis, in contrast to those

 

opr. dubius do not remain in the subcutaneous tissues for
any length of time after injection or penetration. Twohy
(1956) found that most of the larvae of N. braziliensis had
migrated from the skin by 24 hours and that none remained
longer then 48 hours. The random movement of the infective
larvae of N. dubius in the subcutaneous tissues indicated
that these larvae did not respond to the stimuli which direct
the larvae of N. braziliensis to enter the circulatory system.
The recovery of adults from the small intestine after
infective larvae of N. dubius were eXposed to the skin of
mice was no lower than the recovery from subcutaneous
injections. Care was exercised to prevent ingestion of
larvae by the mouse. Since a few larvae were found in the
subcutaneous tissues of mice exposed to infection by skin
penetration, it seemed probable that a minor percentage of
the infective larvae have the ability to penetrate the skin.
Too few larvae survived to determine the pathway of their
subsequent migration. If larvae reached the intestine only
as a result of random migration, it would seem that more
larvae would have accidentally attained the intestine to

develop to adults after subcutaneous injections than skin

5O
penetration because of the greater number in the skin.

The skin is obviously a barrier which can not be
penetrated by most larvae. Twohy (1956) found the percentage
of infection by N. braziliensis was lower after skin penetra-
tion than after Subcutaneous injection. The low recoveries
from the subcutaneous tissues after cutaneous exposure
indicates that it is a greater barrier for‘N. dubius.

Intraperitoneal injection of N. dubius larvae did not
lead to an intestinal infection. Only a few larvae were
recovered after injection into this site. This made it seem
doubtful that subcutaneously injected larvae reached the
intestine via the peritoneal cavity.

Bracket and Bliznick (1949) injected 3. braziliensis
larvae into the peritoneal cavity of rats and reported
recoveries of adults that ranged from 19-55% of the larvae
inoculated. From a similar experiment Simaren (1964) obtained
45% of the N. braziliensis larvae as adults. He also showed
that after being injected into the peritoneum the larvae
migrate through the lungs to reach the digestive tract. It
seems that the peritoneal route of infection reduces the

survival of both N. braziliensis and N. dubius.

CONCLUSIONS

It has been determined in this investigation that the
infective larvae of N. dubius lack many of the abilities
necessary to infect by a cutaneous route. Most of the larvae
can not penetrate the skin of the host but if injected into
the subcutaneous layer can undergo migration. This migra-
tion is not directed towards the circulatory system and thus
the larvae were not carried to the lungs. The larvae were
able to migrate from the lungs to the digestive tract if
artificially introduced into the circulatory system.

A small percentage of the infective larvae possessed
abilities similar to the infective larvae of‘N. braziliensis
and can penetrate the skin and migrate from the subcutaneous
tissues to the small intestine and develop to adult worms,

but the route of migration was not determined.

51

SUMMARY

1. Pepsin digestion of tissues and organs was found
to give low recoveries of third and early fourth stage
larvae, but the efficiency increased as the larvae developed
to adults.

2. Larvae do not migrate outside of the small intestine
after oral infection.

5. When sheathed or exsheathed infective larvae were
injected intravenously, most of the larvae stopped in the
lungs and about 40% of the larvae were able to migrate from
'the lungs to the digestive tract via the trachea and
esophagus.

4. Most of the larvae recovered after subcutaneous
injections underwent lateral migration but only a small
percentage of the larvae were able to migrate to the small
intestine.

5. Larvae injected into the peritoneal cavity did not
survive and only a few larvae were recovered. I

6. About 5% of the infective larvae exposed to the
shaved abdomen of mice were able to penetrate the skin and

migrate to the small intestine.

52

LITERATURE CITED

Baker, N. F. 1954. Trichostrongylidosis--The mouse as an
eXperimental animal. Proc. Am. Med. Assn. 91st.
Annual Meetingzl85-l9l.

. 1955. The pathogensis of Trichostrongyloides
parasites: Some effects of Nematospiroides dubius on
the electrolyte patterns and spleens of mice. Exp.
Parasit.‘&:526-554.

Baylis, H. A. 1926. On a Trichostrongylid nematode from the
cod-mouse (Apodemus sylvaticus). Annals and Magazine
of Natural History. Ser. 9, Vol. XVIII:455.

 

Brackett, S. and A. Bliznic. 1949. Screening large numbers
of new chemical compounds for anthelminthic activity
using infections with 8Nippostrongylus muris in mice.

J. Parasit. 55(1):8

Callizo, P. J. 1962. The freeliving develOpment of
Nematospiroides dubius Baylis, 1926 (Nematoda:
HeligmosomidaII. Master Thesis, University of
California, Davis. As cited by Liu, 1965.

Ehrenford, F. A. 1954. The life cycle of NematOSpiroides
dubius Baylis (Nematoda: Heligmosomidaé). J. ParaEIt.

NE (45:480-481.

Fahmy, M. A. M. 1956. An investigation on the life cycle
of Nematospiroides dubius (Nematoda: Heligmosomidae)
with speciaIIreference to free-living stages. Z.

Parasit. i2;594s599.

Lepak, J. W., V. E. Thatcher, and J. A. Scott. 1962. The
development of Nematospiroides dubius in an abnormal
site in the white mouse and some apparent effects on
the serum proteins of the host. Texas Reports on
Biology and Medicine. §Q(5):574-585.

Lewert, R. M. and C. L. Lee. 1954. Studies on the passage
of helminth larvae through host tissues, I and II.
Journal of Infectious Diseases. Vol. 95. 15- 51.

Liu, S. K. 1965. Pathology of NematOSpiroides dubius.

I. Primary infections in C Ho and WeESter mIce.
Expl. Parasitology 17(2): 12 -l55.

55

34

Roman, E. 1951. Etude ccologique et morphologique sur
les acanthocephales et les nematodes parasites de rats
de la region Lyonnaise. Mem. Mus. Nat. Hist. Nat.,
Paris. N. S., Ser. A, 2001. 2:49-268.

Sadig, S. M. 1964. Obtaining clean infective larvae of
Nematospiroides dubius Baylis (1926). Am. Fed. Vet.
Ma. Q 109 : 783—‘8'47 9.

Schwartz, B. and J. E. Alicata. 1954. The development of
the Trichostrongyle, Nippostrongylus muris, in rats
following ingestion of larvae. J. Wash. Acad. Sci.

25:554-558.

. and . 1955. Life history of
Longistrata musculi, a nematode parasitic in mice.
J. Wash. Acadf Sci. 25(5):128-146.

 

Simaren, J. O. 1964. Quantitative studies on development
of Nippostrongylus braziliensis after different routes
of infection. Proc. HeIfi. Soc. Wash. 5;(2):281-284.

Spurlock, G. M. 1945. Observations on host parasite rela-
tions between laboratory mice and Nematospiroides dubius

Twohy, D. W. 1956. The early migration and growth of
Nippostrongylus muris in the rat. Am. Jour. of Hygiene,
V01. 3, FIG. , T65-I850

Yokogawa, S. 1928. The develOpment of Heligmosomum muris
Yokogawa, a nematode from the intestIne of the wooi-
rat. Parasitol. 13:127-166.

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