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A COMPARISON OF TRANS-TECUMENTAL NUTRIENT UPTAKE

 

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AND IONIC REGULATION IN GUT (Ascaris suum)
VERSES TISSUE (Dirofilaria immitis)
PARASITIC NEMATODES

Date

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presented by

Steven Alan Sedrish

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A COMPARISON OF TRANS-TEGUMENTAL NUTRIENT UPTAKE
AND IONIC REGULATION IN GUT (ASCflI’iS SUUM)

VERSES TISSUE (Dirofilaria immitIS)

PARASITIC NEMATODES

by
STEVEN ALAN SEDRISH

A THESIS

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

MASTER OF SCIENCE

Department of Pharmacology and Toxicology

1989

fix)

.‘. .

ABSTRACT

A COMPARISON OF TRANS-TEGUMENTAL NUTRIENT UPTAKE
AND IONIC REGULATION IN GUT (Ascaris SUUM)

VERSES TISSUE (Dirofilaria immitIS)
PARASITIC NEMATODES

BY

Steven Alan Sedrish

Parasitic nematodes are a very diverse group of
organisms that infect nearly every animal. known. Despite
their diversity very little is known about the mechanisms of
nutrient uptake and ionic regulation in these parasites. The
tegument has been proposed to» play a significant role in
these processes however a means of measuring and comparing
this quantitatively has not been developed.

We designed a system in which segments of these
nematodes were cannulated and perfused in media containing
various radioactive compounds. The perfusate was then
analyzed to determine the extent to which radioactive
compounds could penetrate through the isolated segments.

Using this system we were able to measure mediated

transport processes for many nutrients in Dirofilaria immitis.

These mediated transporters could not be found in Ascaris suum.

We were also able to measure a significant difference in the

movement of ions into these two parasitic nematodes.

To my family for their constant
encouragement and for always being there
when needed.

ii

I would like to thank my advisor, Dr. James Bennett for all
his help. His great ability as an advisor shows in his
guidance and patients with graduate students.

I would like to thank Dr Ralph Pax for all his help with the
design of the experiments and interpretation of the data.
Also I would like to thank the other people in the lab for
all their help.

Finally I would like to thank. my family for all the
encouragement, and a special thanks to my grade school
teacher Ms "mickey" lineal for her help and expertise.

Thanks

iii

Table 9; Contents

 

List of Figures.. ...... ......... .............. . ...... V
List of Tables........ ....... . ....................... VII
List of Abbreviations..... ......... . ................. VIII
Life Cycle of Dirofilaria immitis .......................... 1
Life Cycle of Ascaris suum ..... . . ...................... 5
Anatomy of the Nematode's Tegument ................... 8
Introduction......................... ........ ........17
Medias ..... ... ....... .... ......... ...... ............. 23
Collection and Maintenance of the Parasites .......... 27
Materials and Methods... ....... ‘ ........... ..... ...... 30
Results .............................................. 37
Conclusions and Discussions .......................... 70
Bibliography........ ..... . ....... .......... .......... 78

iv

10

11

12

13

14

15

16

List of Figures

Li f e cycle of Dirofilaria immitis ....................... 4
Life cycle of Ascaris suum. . . . . . . . .................. 7
Diagram of a typical nematodes cuticle..... ....... 10

Diagram of a cross section through a nematode
in the region of the female gonads ........ ........14

Electron micrograph of a cross section through

the tegument of a nematode.......... ..... .........15-16

The various cannulation techniques used.. ......... 33-34

Comparison of the trans-tegumental permeability
to water, glucose and inulin ...... . ..... ..... ..... 38

Uptake of glucose under various conditions... ..... 43

The affect of 1.5mM Thiacetarsamide on glucose
uptake in D. immitis. . . . . . ................. . ......... 45

The affect of external pH on glucose uptake
into heartworm segments ........ . ..... . ............ 47

Glucose uptake into heartworm segments as a
function of temperature ........................... 49

Acetate uptake in D. immitis and A. suum _
segments.........OOOOOOOOOOOOOOOOOOO 00000000000 .0051

Amino acid uptake in D. immitis and A. suum
segments....................... ................... 53

Concentration curves for the uptake into
heartworm segments of leucine, glutamine

and tryptophan .................................... 55-56

Concentration curve for uptake into heartworm
segments for glucose .............................. 59

The permeability of both D. immitis and A. suum
to the drug ivermectin............ ................ 62

17 The permeability of both D. immitis and A. suum to

ionSOOOOOOO..........OOOOOOOOOOO......OOIOOOIOOOO.65

18 Net movement of ions into A. suum expressed in
molar concentrations..............................68

vi

W

Perfusion Medias...... ..... . ....................... 25-26

The permeability to water and glucose expressed in
mM concentrations..... ..... . ..... .................41

Km and Vmax values for leucine, glutamine, and
glucose uptake in Dirofilaria immitis. . . . . . . . . .4 . . . . . . . . . . 60

The permeability of both Dirofilaria immitis and
Ascaris suum to ions expressed in DPM' s. . . . . . . . ...... 64

The net movement of ions into A. suum expressed as
DPM' s O O O O I OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO O 0000000000 67

vii

List 2; Abbreviations

 

A.P.F. ........... ...Artificial perienteric fluid

A. suum . ............ .Ascaris suum

A. viteae . . ............ Acanthocheilonema viteae

B. pahangi . .......... . Brugia pahangi

D. immitis ............. Dirofilaria immitis

IRpm ................. Inorganic RPMImo

RPMIM“, .............. A specific culture media sold by GIBCO

viii

1

Life Cycles

Dirofilaria immitis, more commonly referred to as heartworm,

is an obligate two host tissue dwelling parasitic nematode.
The definitive hosts are the domestic dog and cat however
foxes, coyotes and other wild canids as well as wild cats
serve as a reservoir host for this parasite. The intermediate
host is the mosquito and at least fourteen species of
mosquitos are proven to be effective vectors for this
disease.

Patently infected.dogs'have.circulating'microfilariae in
the blood. These microfilariae show an interesting phenomenon
called periodicity meaning that the microfilaria are present
in the blood in higher concentrations (up to fifty times)
when the mosquitos are actively feeding. This rhythmic cycle
of microfilariae presence in the blood varies depending on
the mosquito host as to whether it is a nocturnal or a
diurnal rhythm.

The microfilariae are picked up by the mosquito host
during a blood meal. While the mosquito is actively feeding
the microfilariae are attracted to the bite wound by a
positive chemotaxis, thus leading to a higher ingestion of
microfilariae than would be expected by the actual

concentration of microfilariae in the blood at the time of

2
the bite. The microfilariae which are actually L.larvae then
pass with the blood meal into the midgut of the mosquito.

The In larvae then burrows their way (within 24 to 36
hours) through the midgut into the malpighian tubules where
they then go through their first ecdysis (or molt) to the L2
larvae. This larval stage then migrates to the abdominal
hemocoel cavity where the second ecdysis occurs to the 13
larvae. This Lgcurinfective stage (for the definitive host)
then migrates through the thorax and into the labium. This
entire process at optimal conditions takes between ten and
fourteen days.

The third stage L3 larvae is than deposited onto the skin
of the definitive host during a blood meal. The 15 larvae
then migrates through the bite wound or burrows through the
intact skin to reach.the subcutaneous tissue‘where the larvae
undergoes a third ecdysis to the 1% larvae (approximately
three to four days post-infection). This 1% than begins a
subcutaneous migration towards the thorax and undergoes a
fourth and final ecdysis to the 15 larvae or juvenile
heartworm (50-70 days post-infection). Then between day 70
and 110 post-infection the juvenile heartworm migrates to the
pulmonary arteries, although the exact route is not known,
it is thought to be through the systemic circulation. Once
in the pulmonary arteries and right ventricle the juveniles

then matures into adult heartworms, the entire process taking

3
between two and four months. The female heartworm does not
become fully developed.and gravid.until about six months post

infection when they begin to shed microfilaria.

ADULT NEMATODES
IN RIGHT VENIIICL!
AND PULMONARY
AI‘I'EIY

’c/“A

   
 

  
 

Fenflhed female reieom

Larvae mature to oduu stage.
microfilariae.

~
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x \ f ’ ~ .
/ '\
// l \
mrecnvr unvu ‘ 2’ ' X \ nosomro moms
us: to uosr random-us
u nosomro mos am noon mm

V

Microfilorioe develop into
infective Std-me larvae.

Figure 1: Life cycle of D. immitis (Ivene, V.R. 1981)

5
Ascaris suum is a one host gut dwelling parasitic

nematode. The life cycle is direct with the domestic pig as

its host. Although there are similarities between the porcine
ascarid (A. suum) and the human ascarid (A.‘Iumbricoide3) and

each are capable of growing to full maturity in the other's
host, they are still considered to be separate parasites.

Adult ascarides live in the small intestine of their
host where they are capable of laying huge numbers of eggs
per day (up to 200,000). The eggs are passed out with the
feces as fertilized.but un-embryonated eggs. Once in the soil
they embryonate and become infective second stagealq larvae.
This process takes approximately two weeks to occur.

The L2 larvae are then swallowed by the host and hatching
takes place in the stomach and small intestine, stimulated
by temperature, Pcoz,ij, bile salts, and the presence of a
non—specific reducing environment. The 12 larvae then
penetrates the lumen of the small intestine and is picked up
by the portal circulation and brought to the liver. Once in
the liver they migrate throughout the liver causing their
characteristic pathology of hepatic scarring.

The larvae then travels through the vena cava to the
heart and then through the pulmonary artery to the lungs.
Once in the lungs the L2 larvae molts to become the L3 larvae
and either becomes lodged in the alveolus or travels to the

somatic tissues. The larvae that are lodged in the alveolus

6
eventually breaks loose from the alveolar capillaries into
the alveolar space where it actively migrates up the bronchi
into the trachea and is swallowed.

The lg larvae then goes through two more ecdyses in the
small intestine to become theZDSlarvae or immature ascarid.
This immature ascarid will then mature to become a mature
ascarid, the whole process taking approximately six weeks
from ingestion of the infective egg to the paSsing fertilized

eggs in the feces.

ADULT NEMATODES
IN SMALL INTESIINE

   
  

  
 

533, hatch. Larva. {gnaw Eggs are laid in mail intestine.

trachea: migration and
mature to count stage.

HOST moans @}@}

mrecrtv: zoos L.-

EGGS m FECES

Larvae devet'op to int‘ecive
2nd stage in eggs.

Figure 2: The life cycle of Ascarissuum. (Ivene, V.R.l981)

8

Anatomy of the Nematode's Tegument

The phylum Nematoda is a very diverse group of animals,
consisting of many thousands of distinct organisms. As a
result of this diversity it is impossible to talk about the
anatomy of all nematodes accurately, but rather what follows
is. a. general description. of ‘the non-existent "typical"
nematode tegument.

The nematode tegument can be divided into three main
structural components or layers, the cuticle, the hypodermis
or epidermis and the muscle. Each of these layers can be
divided into separate layers and in fact each of these
divisions can be divided structurally and functionally into
further divisions. However for this discussion we do not need
to and will not go into as much detail as is possible.

The outermost layer of the tegument is the nematode's
cuticle. The cuticle is divided into three main layers that
are both structurally and presumably functionally distinct.
The outer most layer or cortical layer, a medial layer or
matrix layer, and an inner layer adjacent to the hypodermis,
the basal layer.

The cortical layer needs to be divided further into an
outer and an inner cortical layer. The outer cortical layer
is considered to be a trilaminate plasma membrane

(plasmalemma) of approximately 100 A in thickness. The outer

9
layer also contains, as viewed on an electron micrograph a

"fuzzy" coating. This coating is thought to be the antigenic
component of the tegument. Ascaris suum emits the most

antigenic protein known to man.

This layer also contains lipids, proteins and
carbohydrates. It is formed directly from the cytomembrane
of the endoplasmic reticulum.

The internal cortical layer consist mainly of a collagen
like protein. This layer is also rich in enzymes, RNA,
ascorbic acid, ATP, and acid phosphatases and is presumably
metabolically active. This ‘layer is formed from
differentiated hypodermal cytoplasm.

The matrix or middle region of the cuticle is a
homogenous layer'that‘varieslgreatly'between.nematodes in the
degree of organization. This layer is also rich in collagen
like protein forming rods and canals (at 75°tx>the long axis
of the nematode). To various degree's these rods and cones
form the beginning of a lattice network.

The basal layer consists mainly of the same collagen
like proteins forming a network of rods and canals. The first
layer runs approximately 135° to the rods in the matrix and
together with subsequent layers form a strong lattice that
gives the nematode shape and allows for the build up of a
high internal hydrostatic pressure without the nematode

actually exploding

10

EXTERNAL CORTICAL

MES: 11 N LAYER

 

 

 

 

LAYER
INTERNAL
CORTICAL

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LAY R [

 

 

 

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Figure 3 :
1971)

Diagram of a typical Nematode's cuticle (Bird,

11

The next main layer, just below the lamella of the
cuticle, is the hypodermis (or sometimes called the
epidermis). The hypodermis is a cellular or syncytial layer.
The main function of the hypodermis is thought to be the
secretion of the cuticle.

The most notable characteristic of the hypodermis is
that the protoplasm of these layers expands into the
pseudocoel cavity. These protrusions form four "cords"
containing all the nuclei of the hypodermal layer. Once the
adult nematode is formed, after the last ecdysis the number
of nuclei remains constant.

The cords are divided into two midlateral, one
midventral, and one middorsal cord and extend the entire
length of the nematode. The middorsal and midventral cords
contains the dorsal and ventral nerve trunks, respectively.
The two lateral cords contain the lateral canals of the
excretory system

The hypodermal layer is very metabolically active and
contain lipids, mitochondria, golgi apparatus, and enzymes.
The four cords are especially rich in mitochondria and
endoplasmic reticulum.

The third main layer of the nematode's tegument is the
muscle layer. The muscle layer consists of longitudinally

obliquely striated fibers arranged in bands. The muscle acts

12
to propel the nematode by contraction against the fluid
filled pseudocoel and thus produces a hydrostatic pressure.

The muscle consists of two main types of fibers. Thick
fibers (23nm diameter) and thin fibers (Snm diameter). The
thick fibers contain myosin while the thin fibers contain
actin and contract. in. a similar fashion as vertebrate
striated muscle through actin/myosin interactions.

The base of the muscle fiber, containing the contractile
fibers, are against the hypodermal layer. The side of the
muscle fiber with the nucleus is towards the pseudocoel. In
addition each muscle fiber has a long slender arm like
projection. This projection extends to the dorsal or ventral
nerve trunk. This is different than the normal situation
where the nerve projects to the muscle.

The pseudocoel or sometimes called pseudocoelom
although not a part of the tegument is worth mentioning at
this point. The pseudocoel is derived from the embryonic
blastocoel rather than from an invagination into the
endomesoderm. Therefore the jpseudocoel does not have a
mesoderm lining. The function of the pseudocoel is as a
hydrostatic skeleton.

The pseudocoel is filled with a cell free liquid. Any
nutrients that are absorbed are presumably transported to
this fluid. This liquid contains electrolytes, proteins,

albumin, globulins, hemoglobins, enzymes, fats, and

13

carbohydrates. The most noticeable fact concerning this fluid
is that it contains a lower concentration of 01' then would
be expected from the concentration of positive cations. This
electrostatic difference is made up by anionic organic acids.
This low Cl' concentration will be discussed again in this
paper in more detail and might be a very important fact.

Another fact about this fluid is that it can maintain a
high hydrostatic pressure against the body wall of the
parasite. This pressure has been reported to be between 70-

120 mm Hg in Ascaris suum.

Figure 4:

14

protoplasmic core of muscie cell

dorsal chord7 [-a’orsal serve

/—7: \/ “um

. ...lrl

    

   
   

 

the region of the female gonads. (Noble, E.R. 1974)

Diagram of a cross section through a nematode in

15

Figure 5: Electron micrograph of a cross section through the

tegument of an ascaris, (Aggarwal, S. Professor of Zoology,
Michigan State University ).

16

 

 

17
Introduction
Parasitic nematodes are a very diverse group of

organisms. They parasitize almost every vertebrate and

invertebrate organism known. The two nematodes, Dirofilaria immitis

and Ascaris suum, used in these experiments are of great
economical importance in today's society.
Dirofilariaimmitis has been estimated to infect anywhere from

0 to 75% of the canine population in any one place, depending
on the climatic conditions. In Ingham County, Michigan 7.5%
of the county seizures (dogs picked up by the county animal
control agents) were tested ‘positive for circulating
microfilaria (approximately 50 dogs were tested). It is also
estimated that.an additional 30% more would test.positive for
occult infections (meaning that they are infected but the

female is not shedding microfilaria).
Ascaris suum is also a very important disease in today's

society. It has been estimated that between 20 and 100% of
the hogs in the US are infected at any one time. The American
pork producers raise 700 tons of Ascaris eggs and 5,000 tons
of adult Ascaris yearly (Krull, 1969). These parasites are
raised accidentally by consuming feed that would normally be
utilized by the porcine host.

Despite the diversity and importance of these parasites

little is known concerning the method of nutrient uptake. The

18
first thing that becomes apparent when one starts to review
the literature in this field is that for every nematode there
appears to be separate mechanisms involved. This fact has
lead to a great deal of confusion in the past since no one
generalized rule is available by which all these parasites
can be described.

As little as fifteen years ago the tegument of the
nematode was thought to be only an outer covering giving
shape and integrity to the parasite. In a review by Pappas
in 1975, on nutrient uptake in all parasites, the cuticle of

nematodes was not even mentioned. In that particular review
Ascaris suum was used as an example of a typical nematode and

all that was talked about was absorption through the gut. As
a result of these early reports very little research was
performed until recently.

Most of the literature looking at trans-tegumental
nutrient absorption in Ascaris suum used cuticle preparations

that were "stripped" of the muscle (Fetterer, 1986). In these
preparations the teguments were normally inverted and then
stripped of muscle either physically or with some enzymatic
digestion. Although these preparations remained presumably
intact by the fact that inulin remained impermeable, it seems
unlikely that the tegument would have behaved normally in

such a state. These experiments all showed results which

19
suggested that the tegument of the Ascaris remained
impermeable to macromolecules.

Flemming in 1984 conducted a series of experiments using
whole intact Ascaris suum in which he cannulated at either end

and was able to perfuse the pseudocoel cavity. He reported
that a significant amount of glucose and 3-O-methyl glucose
was able to enter the parasite by a trans-tegumental route.
He postulated that possibly the reports of this barrier being
impermeable might be incorrect. However when one actually
looks at the quantity of glucose entering the parasite and
compares it to how much enters through the gut this quantity
is insignificant. Also he did not report any data suggesting

any process other than simple diffusion occurring.

Many studies have been performed on the gut of Ascaris

mmnn (Castro, 1969; Beames, 1971) describing the gut as the

main site of nutrient uptake. These reports have demonstrated
specific and saturable processes for absorption, not unlike
the gut of higher organisms.

Rutherford in two papers, (1974, and 1977) using the
nematode Mermis nigrescens reported saturable transport
mechanisms in the tegument for glucose and certain amino
acids. In these experiments it was shown that the glucose
transport was saturable and concentration dependent but not

energy dependent or coupled to a sodium transporter. This

20
uptake was shown to be sensitive to 105M phloretin and 104M
dinitrophenol. In the same report he showed the absence of
a transporter for trehalose, the major sugar moiety in this
parasite's natural host.

His reports stated that this transport mechanism
occurred only in the 14 day old larval stage and not in the
thicker cuticled 21 day old larvae. This suggested that this
mechanism exists on the hypodermal side of the cuticle.

The transport of molecules across the cuticle was in the
past described strictly on the basis of size or partition
coefficients (Fetterer, 1986).

Many reports of filarial nematodes state that their
tegument is permeable. Once again however, no one rule exists
for all filariids. Court, in 1983, looked at transport of

3'5'-cyclic adenylate monophosphate (cAMP) and isoproterenol
into Litomosiodes carinii and Acanthocheilonema viteae . This paper

stated that both parasites took up cAMP but only A. viteae

took up isoproterenol.

Brugia pahangi however was shown to have a correlation of

.99 with A. viteae when looking at the uptake ‘of several non-
electrolytes (Court, 1988) .Several experiments were performed
on B. pahangi and D. immitis, by Chen and Howell in 1980, 1981,

and 1983. These experiments although very simple in their

21
design have had a great impact on the thinking on trans-
tegumental uptake.

These experiments consisted of incubating parasites in
petri dishes, where the parasite‘s anterior, posterior, and
middle regions could be exposed to different environments.
Two facts came from these experiments. First, by incubating
the anterior and not the posterior region of the parasite
with radioactive tracers, they were not able to detect any
radioactivity in the posterior region of the parasite. This

they demonstrated for both D. immitis and A. viteae and

postulated that no movement through the gut, in an anterior
to posterior direction, occurred.
This was a very interesting observation but the

conclusion, although now considered to be true by this

author, was inconclusive. This is because ”IWNO they showed
no oral absorption of trypan blue dye, which does occur #7

VWO. This signifies that their system was not perfect and
needed to be adjusted until concurrent information could be
obtained from fltwmb and filwWO data.

The second conclusion from these experiments is that
they showed specific trans-tegumental transporters for

several macromolecules. Included among them was glucose,

adenosine, and several amino acids. Glucose uptake into £1

immitis was found to be stereospecific in that l-glucose was

22
not taken up. Also in D. immitis the nucleic acid precursor
thymidine and the disaccharide sucrose was not shown to be
taken up. In B. pahangi glycine was shown to be competitive

with methionine, valine, and phenylalanine: and to be

noncompetitive with arginine.

It appears that D. immitis, a filarial parasite, shows the
most promise for the measurement of trans-tegumental uptake
mechanisms. It also appears that A. suum, the classically used

nematode, has the most impermeable tegument.

23
Media
Several media are utilized in my work with regard to
both incubations and the perfusion of parasites. To aid in
the understanding of this paper the different media will be
described. However for the specific media used in each
individual experiment refer to the experimental procedure

section.
Dirofilaria immitis has in the past been difficult to keep

viable in culture. For this reason several variations on the
culture media for heartworm (Heartworm media) were tried
until a satisfactory media was found. The heartworm media
used to keep heartworms alive in culture contained the
following RPMImw with 5% Calf supreme (a fetal calf serum
replacement manufactured by Gibco Company) also this media
contained 50 mg/L gentamicin sulfate and 2.5 mg/L
amphotericin B. This media was adjusted to a pH of 7.4, with
the use of 20mM hepes buffer.

The RPMIWO contains 10mg/L phenol red indicator. This
indicator at a pH of 7.4 is a bright red color, while in a
more acidic solution it turns yellow. This color change
allowed us to know when the media needed to be changed due
to the acidification of the media by the parasite. The media

was changed approximately every three days.
The best culture media for Ascaris suum is still a matter

of some debate. There are two main media used for this

24
purpose, Donahue's media and A.P.F. (see table No. 1). The
main differences in these media are that both contain Hepes
as a buffer but Donahue's media also contain 10 mM HCOf as
an additional buffering system. The other main difference is

that A.P.F. contains 128 mM acetate, this acetate is added
under the belief that Ascaris suum are capable of utilizing

this acetate for energy. Other differences, although slight
are shown in table 1. Inlall experiments, unless specifically

stated differently Donahue's was used as the culture media
for Ascaris .

One other media used was inorganic RPMI (IRpm) . IRPM, is
a solution to simulate the inorganic ions in organic RPMI
(RPMImo) without most of the organic added. However IRpm does
contain 11.1mM glucose (except in the experiment looking at
dose response curves for glucose uptake) and 20 mM Hepes as

a buffering system.

25

Table 1: Shown here in millimolar concentrations are the
inorganic and organic constituents of the three commonly used
medias. Also shown is the pH's of the medias

Imm, A.P.F. Donahue's
Inorganic
Na+ 102.7 131.9 121.0
K+ 5.36 24.0 24.0
Ca“' 0.44 11.8 1.0
Mg++ 0.41 9.8 5.0
c1’ 107.36 71.1 151.0
No; 0.88 - —
P0,;3 5.64 - 0.5
sof 0.41 - 5.0
Hoof - - 10.00
NH{‘ — - 0.75
Organic
Acetate - 128.0 -
Glucose 11.0 10.0 27.0
Hepes 20.0 5.0 5.0
pg
pH 7.4 7.0 7.0

 

27

Collection and Maintenance of Specimens

Dirofilaria immitis were collected from either random source,

naturally occurring microfilaria positive dogs obtained from
the local pound or from an NIAID supply contract 1. In the
former case the dogs were tested for the presence of
microfilariae by filter occlusion testing of venous return
blood obtained from the cephalic vein. If the dogs tested
positive for microfilariae the dogs were euthanized by C02
asphyxiation in an approximately 90% C02 atmosphere (1986
Report of the AVMA Panel on Euthanasia). The heart and lungs
were quickly removed and the parasites (heartworms) dissected
out. The parasites were then put into a solution of sterile
RPMI 1640 with antibiotics (see media section) and,
transported to the laboratory.

The parasites from the NIAID supply contract were
obtained in a similar fashion and shipped in RPMI 1640 by
overnight express to the lab. Once at the lab these parasites
were treated in the same manor as the parasites obtained from
random source animals.

Prior to placement into incubation flasks, the parasites

were rinsed with a sterile saline solution and then placed

 

l Filarial materials used in these experiments were donated
by an NIAID supply contact (AI #02642), US-Japan cooperative
medical program.

28

into sterile heartworm media (see media section). The media
was adjusted prior to the addition of worms to a pH of 7.4.
The media was kept at room temperature until several hours
before the experiment when they were put into a 37°
incubator. The worms were rinsed with sterile saline and put
into fresh media approximately every three days (or as
needed) as indicated by the acidification of the media by the
parasites.

The heartworms were able to survive in this media for up
to one month. The length of the incubation did not affect the
experiments in which we were looking at organic transport,
however, inorganic permeability did increase significantly
after one month of incubation. Therefore all experiments on
inorganic transport were done within a week of euthanizing

the animal.
Ascaris suum were both obtained from the local abattoir

or from lab raised and infected.hogs (porcine). In both cases
the animals were sacrificed by electrical stunning followed
by exsanguination. The parasites (ascarides) were quickly
removed from the small intestines and placed into Donahue's
media (except in those experiments where a different media,
A.P.F., is specifically stated). The parasites were
maintained at a constant temperature of 37‘C and transported
as quickly as possible back to the laboratory. Once at the

lab the parasites were rinsed with and placed into fresh

29
media. The media was maintained at a temperature of 37”C and
a pH of 7.0 and changed daily. The parasites were able to be
kept in the media for up to one month but were used in all

cases within one week.

30
Materials and Methods

The radiolabeled chemicals were purchased from the
following sources. The 14C Leucine (337.1 mCi/mmole) , Arginine
(270 mC/mmole), Asparagine (129 mCi/mmole), Glutamine (250
mCi/mmole), and glucose (220 mCi/mmole) was purchased from
ICN radiochemicals. The 1"C tryptophan (119 mCi/mmole) and
Acetate (94 mCi/mmole) was purchased from NEN research
products. The radioactive ions ZZNa“ (49.65 Ci/mmole, adjusted
for decay), 36c1‘ (1062 Ci/mmole), and “Ca“ (3.90 Ci/mmole)
as well as the 3H H20 also came from NEN research products.
The 3H (11.1 Ci/mmole) labeled Ivermectin was a gift from the
Upjohn company. 1

The RPMIWO and the Calf Supreme was purchased from

GIBCO Co. All other chemicals were purchased from Sigma

chemicals.
In the case of Dirofi/aria immitis the parasite is allowed to

adjust to 37°C for several hours before the start of the
experiment. After this equilibration time the parasite is
then placed into a petri dish containing 1m, also at 37°C.

Polyethylene tubing (PE-50) is heated over a soldering
iron and pulled out to a fine tip. This allows the tubing to
be inserted into the parasite's body cavity.

The heartworm is then cut into a segment of length
between .5 and 1.5 cm's. When the parasite is cut the

internal organs are expelled due to the internal turgid

31
pressure. This expulsion can be used to judge the viability
of the parasite and if it did not occur the parasite was not
used for an experiment.

Once the heartworm is cut into a segment the uteri and
gut is removed by gently pulling it through the body cavity
with tweezers. Special attention is paid so as to not damage
the body wall. Both sides of the segment were then cannulated
with the PE-50 tubing prepared with a fine tip. Both ends are
then sealed with a cyanoacrylate glue (Figure 6).

In preparing the segment there is a contraction of the
body musculature which will again relax when the segment is
perfused. To standardize the measurement of the length of
segment the length was measured after cannulation and before
any'perfusion‘wasjperformed. Once antaccurate length has been
taken the segment is tested by pushing A.P.F. through it with
a syringe. If any leaks are detected the segment is

discarded.
For Ascaris suum two separate methods of cannulation are

used, one for normal segment preparation and a separate
method for an inside out segment. In both cases the parasite
is placed into a petri dish containing IRpm or Donohue's
media depending on the experiment. The ascarid is then cut
using a razor blade and again there is an expulsion of the

internal organs that can be used as an estimation of the

32

viability of the parasite. The intestines and the uterus are
then removed similarly to the heartworm.

In the outside out (Fig 6) preparation a length of PE-
90 tubing is prepared with a 1 cm length of silastic tubing
placed around the tip. The end of the PE-90 tubing with the
silastic tubing tip is inserted into the ends of the parasite
approximately % cm. Both ends are then tied closed with 4-0
silk sutures and sealed with cyanoacrylate glue (Figure 6).

In the inside out ascarid preparation one piece of PE-
90 tubing is inserted completely through the entire length
of the body of the cut and cleaned segment. The far end is
tied off with a suture and the body wall of the parasite is
feed back over itself. The second piece of tubing is then
inserted and tied off. Then both ends of the segment are

sealed with cyanoacrylate glue (Figure 6).

33

Figure 6.'Phis figure shows the various cannulation techniques
used. A: For a heartworm. B: For an outside-out ascarid. C:
For an inside-out ascarid.

35

Once the segment is cannulated, the integrity of the seal
is tested by perfusing solution through the segment while it
is still out of the bath and visually checking for leaks. The
integrity of the seal is also tested when the perfusion starts
since there is air in the system the appearance of bubbles in
the bath signifies a "bad" seal.

One end of the segment is connected to a perfusion pump
that is set at a pumping rate of %ml per minute. The other
end of the segment is connected to a fraction collector set
to sample at thirty second intervals.

The segment itself is placed into a petri dish containing
15 ml media. The media is either Immp, A.P.F., or Donohue's
media depending on the individual experiment. The petri dish
is placed into a water bath were the temperature is kept
constant at 37°C, unless otherwise specifically stated.

Radioactive tracer is then added to the 15ml bath. When
working with 1(‘C or 3H 4uCi of radioactive tracer is added.
When working with other radioactive isotopes less tracer is
used.

The perfusion is started and as stated before the air in
the line provides a indicator if the segment is intact. The
perfusion is allowed to run for ten to fifteen.minutes (unless

stated) until the fraction collector is turned on. The

36
perfusion is then carried out for the time specified in the
experiment.

The %ml fraction is then added to 6-8ml aqueous counting
cocktail. The entire amount is then counted for radioactivity
with a Beckman scintillation counter.

Once the number of DPM's are known, the actual amount of
radioactive solute going across the tegument can be calculated
from the specific activity of the solute. Then from the ratio
of radioactive to known radioactive solute, the amount of

total solute can be calculated.

37

BQEELEE

Our first consideration was, to'determine if the process
of cannulation damaged the parasite's tegument was damaged.
To look at this, a large molecule that is considered to be

14

impermeable, in intact membranes, was used. 4uCi of C

labeled inulin (mw > 2000) was added to 15ml 0f3me and both
Ascaris suum and Dirofilaria immitis were perfused with APF at 37°C.
As can be seen in figure 7 the membranes of both cannulated
preparations were impermeable to inulin. From this result it
can be assumed that the tegument of these preparations were

not grossly damaged.

38

(n
O
O
0

4,000

M'Otii'titi‘
I‘d
'-—~t

3,000

 

 

2,000
a D. immitis

m A. suum

DPM/cmZ/min

 

”+1’H'0thleH-tlt-H4h

 

 

1,000

 

 

 

 

- +mf+mlo

l .. .

{RX}? 1192:!— ‘ .
. . 4—4
Water Glucose lnulin

 

O

" = P<.05 (T Test)

Figure 7: Comparison of the trans-tegumental permeability to
water, inulin, and glucose.

39

The next experiment was to look at the diffusional
properties of these segments. 4uCi UIIQO was added to 15 ml
In..." and perfused with A.P.F. at 37°C. In a similar experiment
4uCi 1"C Glucose was added instead of the 3H water. The
results of both sets of experiments can be seen in figure 7.
From this figure it can.be observed.that the ascaris tegument
represents a much greater barrier to both water and glucose
than does the heartworm tegument.

The heartworm tegument was approximately 18 times more
permeable to water and 34 times more permeable to glucose
than was the ascaris tegument (Table 2). The fact that the
ratio 'was not constant indicates the poSsibility of a
transport mechanism for glucose in the heartworm tegument at
37°C. If glucose was being moved across the tegument by
simple diffusion you would expect that the glucose would
follow along with the water and.the ratio would have remained
constant.

Although in figure 7 the DPM's for water and glucose
movement into heartworm and ascaris segments was
approximately the same as that for water, the actual movement
of solute (both radioactive and non-radioactive) was
drastically different when calculated, because the ratio of

unlabelled (cold) to labelled (hot) was much greater for

40
water than glucose. From this it can be assumed that the
heartworm is permeable to water but is less, to be shown

later, selectively permeable to other molecules.

41

Water
D. immitis 3.45x10'1 1 0.70x10'1
A. suum 0.19x10'1 1 0.06x10'1
Ratio 18.2

Table 2: The permeability to water
mM 1 SE concentrations.

Glucose

7.58x10‘8 1 0.77x10'8

0.22x10'8 1 0.041(10'8

34.0

and glucose expressed in

42

To decide if the uptake of glucose was a transport
dependent process the same experiment was conducted with
glucose both at 37°C and 4°C. Also in the same experiment the
effect of removing the glucose from the APF, which is
normally in 10mM concentration, was performed. Removing the
glucose from APF made the inside of the segment devoid of
glucose. The experiment was run with and without 10mM glucose
in APF at 37°C and with 10mM glucose in the APF at 4°C. If the
process was controlled by a transport mechanism there should

be a significant decrease in uptake at 4°C.

43

10:
:1

mol/cm2/min X10”8
0

 

 

 

 

 

 

47///////////////////a;

T [:1 = 37°C
' - T = 40C
-0mM Glu in APF

D. immitis A. suum

0.1

* = P<.05 (Paired t Test)

Figure 8: Uptake of glucose under various conditions.

44

Shown in figure 8 are the results from the experiment.
There was a significant decrease in glucose when the glucose
was removed from the inside of the preparation in the
heartworm, while in ascaris there was no effect. Likewise
there was even a further decrease in glucose uptake in
heartworm, both significant from control and from the 0 mM
glucose when the temperature was decreased to 4°C. This
decrease again did not occur in ascaris.

Several known organic and inorganic poisons were
examined for their ability to inhibit the transport of
glucose into the heartworm segment. Among these were 104M
Ouabain, 10'5M Thio-D-glucose, 10'3‘KCN, 10'3 Sodium azide, 10'
4 antimony potassium tartrate, 10'5 Phlorizin, 10'3 Phloretin,
and low sodium in the external media.

The only compound that had a significant and repeatable
effect on glucose uptake was CaparsolateR (Thiacetarsamide).
Thiacetarsamide is an adultacide for heartworm and is
presently the drug of choice for the disease. Shown in Figure

9 is the results from one experiment looking at 1.5mM

thiacetarsamide.

45

 

10,000?

 

 

:: I
8,000 1:
1‘;
0-

4|-

«>-
6,000 1: ,
:— I' e
4,000 «I

 

 

I

 

.1

2,000;

__

DPlvl/min/cm2 Surface Areo

IVTTj'I5Y

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1t-
0 ‘P

l T *1

Control Cop 30 60 Wash 30

. = p<.05 (Paired r Test) Time (mins)

Figure 9. The affect of 1.5mM thiacetarsamide on glucose
uptake in Dirofilaria immitis .

46

As can be seen in figure 8, thiacetarsamide rapidly
decreased the uptake of glucose into a heartworm segment,
which was significant within 30 minutes. The effect of the
arsenical was reversible however at 60 minutes.

Since pH is an important variable in the transport of
nutrients across biological membranes we analyzed its affect
on glucose uptake. Iwm: is normally on the outside of the
worm in this system, and has a pH of 7.4. APF, which is
normally on the inside of the heartworm in this system, has
a pH of 7.0. Experiments involving the changing of pH's of
both medias were performed and the results can be observed
in figure 10. Changing the pH of the external environment,
in either direction, caused a significant decrease in glucose
uptake. Changing the pH of the internal environment (APF)
from 7.0 to either 6.0 or 8.0 had little if any effect on

glucose uptake.

47

N
UT

O
O

UT
0
t—l +4l49—VH eifi‘l- 4 t—Ho 44F+H5flfl¥i

   

Percent of control
\l
()1

25

6.5 7.4 810
x = P(.05 (T Test)
pH

Figure 10: Affects of external pH on glucose uptake in
heartworm.

48

The obvious effect of temperature on glucose uptake when
the temperature was decreased from 37°C to 4°C led us to try
to quantify the affect. 4pCi 1('C glucose was added to 15ml
Iv“ and several heartworm segments were perfused with APF.
The temperature of the water bath was set at 430 to start
with and raised approximately 5 to 10°C every ten minutes. In
similar experiments inulin was used instead of glucose to
determine to what point the temperature could be raised
without damaging the tegument membranes.

As is shown in figure 11 ‘there ‘was_ a ‘temperature
dependent increase in glucose uptake. However a Tm, could not
be determined because the transport of glucose was still
increasing at a temperature where the tegument began to
break down i.e., between 50 and 60W: inulin began to pass

through the membrane into the APF perfusate.

49

 

 

5 x 10—7
.l— A
Q?
E .
m\ A O
E A C
o
E 1 10‘7 .
g x 1' A .
2 C
B
3 e
a) A
(D
8
3
5 I
1X 10— i 5 i c % ¢ ¢ : c : : : t : fl]
0 25 50 75
Temp (0")

Figure 11: Glucose uptake into heartworm segments as a
function of temperature.

50

Lately there has been much interest with acetate in
nematodes. Several investigators have recently shown that
ascaris are capable of utilizing acetate as an«energy source.
It was previously thought that acetate was strictly an
excretory product that was eliminated by the parasite. With
this in mind and with the information previously gathered on
the absence of a glucose transporter in ascaris tegument the
experiments were performed to determine the presence or
absence of an acetate transporter in these parasites.

As was shown in the media section Ikpm normally does not
contain acetate while APF contains 128mM acetate. 4uCi of 1"C
labeled acetate as well as 1mM cold acetate was added to the
incubation bath of 15ml Iwm and the segments were perfused
with APF at 37°C and 4°C. The results of these experiments are

presented in Figure 12.

51

...L
O
; 1 I

 

mol/cmZ/min X IOTB

 

 

 

 

 

[:1T = 37°C
-T — 4°C
1 r , A,

D. immitis A. suum

#- : P<.05 (Poired T Test)

Figure 12: Acetate uptake into Dirofilaria immitis and Ascaris suum
segment.

52

It can be seen in figure 12 that no acetate was
transported into the ascaris segment even in-the presence of
a large concentration gradient. The heartworm segment was
however able to transport acetate into the segment. This
transport was significantly decreased at.4PC. It should also
be noted that the radioactive acetate could be displaced from
entering the preparation if nonradioactive acetate was added.
As noted before this transport took place against a
concentration gradient of over one hundred fold and still
occurred at a very high rate, comparable to glucose.

Four amino acids were picked to represent the four
different "RF groups which are, nonpolar and uncharged, polar
but uncharged, polar and negatively charged, and polar and
positively charged. Leucine, asparagine, glutamic acid and
arginine were used respectively. 4uCi of 1("C labeled amino
acids were added to 15 ml of RPMImo which contains unlabeled
amino acids. The segments were than.perfused.with.APF and the

perfusate counted for radioactivity. This was done in both

Dirofilaria immitis and Ascaris suum .

53

 

 

 

 

 

 

 

 

20]—
‘3 10:; l
.0 ‘- ,1 1*
g: . 1 .
E
Q A. suum
E m T = 37°C
E * E’ZZT = 4°C
2 D. ImmitlS
O
CTT - 37 C
1.0 :— T -T _ 4°C
" *
Leu Asn Olu Arg

. = P < .05 (Paired T Test)

Figure 13: Amino acid uptake into Dirofilaria immitis and Ascaris
suum segments.

54

Figure 13 shows the results from these experiments. As
can be seen on the graph the bars for ascaris are missing
this is Ibecause ‘under' these, conditions radiation above
control was not detected, signifying that the amino acids
were unable to enter the ascaris preparation.

This figure also shows that a significant amount of each
amino acid was transported into heartworm segments under
these conditions. Transport significantly decreased when the
temperature was decreased to 49C. This decrease was
reversible when the temperature was raised back to 37”C.

The next set of experiments were designed to determine
to what extent the transport of nutrients into heartworm
segments was concentration dependent. Three amino acids as
well as glucose were used. Amino acids used were leucine (an
uncharged and non-polar amino acid), glutamine (an uncharged
and polar amino acid), and tryptophan (also a non-polar and
uncharged but somewhat larger amino acid). All three amino

acids used were 1['C labeled as was glucose.

55

Figure 14;A,B,C: concentration curves for the uptake into
heartworm segments of leucine, Glutamine and Tryptophan. The
------ line represent the actual experimental values
obtained. The . ..... line represents the calculated
diffusional component of transport. The ———-—- line
represents the corrected transport.

Uptake (mol/cmz/m'tn) ‘ 10-10

Uptake (moI/cmz/min) x 10-9

-8

Uptake (mol/min/cmz) x 10

“NUEUICDVGILO

0

56

 

 

 

 

 

 

 

 

+
“ . T ............................................................ I Actuo'
t I.) 1 Corrected
1h
4)-
“ 7 ‘ Diffusion

4% L A n l 1 I 1 l . 1 1 L l

T T I V V T I 1' V I Y Y I Y Y I’ I V V V ]

Leucine (mM) 0 .5 l O
....................... Actual
Corrected
Diffusion
A 1 l l t l l l 1 l l 1 l l L 1 l 1 1 1 1 . t
I ' T I I I ' T ' I r T ' T l ' l ' l i i ' i T i ' T 7 i i ' i
2 3 4 5 6 7 8 9 10

J
I I

e
G
n
O
‘
D 1
p
)—
4!-
y.
p.
u.
t-
y-
.—
P'
y.

A

 

.-'
.-
."
."
."
.-
."
.I'
.“
_e

."
.-
,u
.a
'0
.-'
-
4"
,-
,o
e
."
.0'
,.
.l'
."
.l'
,o

.¢'
a'.'
o"
.“
."
.I‘
..
.a
.-
.-'
."
..
."
.0'
,n
.

Trvotoohon (mM) 0 OS 0 10 (1.15 0 9O

57

These three graphs, Figures 14:A,B,C show the results
from the amino acid experiments. Figure 14:A shows the
results for leucine. The ------ line shows the actual
experimental values obtained. The shows the values
that were calculated for how much of the actual transport can
be attributed to simple diffusion. The --——- line shows
what is left of the actual experimental data when the
diffusional part is subtracted. From this a value for the K,"
(or half maximal concentration) was calculated to be .68mM
and a value for the Vmax (or maximum rate of transport) was
calculated to be 2.72x10'8 (shown in table 3).

Figure 14B shows similar graphs for glutamine. The
values for K,“ and me are shown in table 3. As is shown in
table 3 there is a significant variation in Km and V"m values
between the various amino acids. This would be expected to
vary depending on the individual need for each nutrient.

The third graph, figure 14C, shows the experimental
values for tryptophan. The amount transported into the
heartworm segments was linearly related to concentration.
Addition of large amount of unlabeled tryptophan was not able
to displace any of the labeled tryptophan from entering the
tegument. Also when at the end of the experiment the
temperature was decreased to 4°C no decrease in uptake

occurred. From this data and as can be seen on the graph

58
there was only a diffusional portion of the transport and no
active process that can be measured.

This next graph, Figure 15, shows a similar graph for
the concentration dependency of glucose on uptake. This graph
shows clearly, the:diffusional and active transport.parts for
the experimental values. Values for K"1 and for Vmax were

obtained and can be seen in table 3.

59

 

 

 

i "[ ACTUAL

(I)
9 5.".
2; L DIFFUSION
NE -
e 4-~
t: I
0 q.—
E
:35 2+- CORRECTED
3 _

O 1 1 1H

10 12

 

Figure 15: The concentration curve for uptake of glucose into
the heartworm segments. The ------ line represent the actual
experimental values obtained. The ..... . line represents the
calculated diffusional component of transport. The

line represents the corrected transport.

60

 

Nutrient K... (M) v",ax (mM/min/cmz)
Glucose 0.68 2.72x10'8
Glutamine 0.14 2.87x10'9
Leucine 0.014 2.73x10*°

Table 3: K"I and Vmax values for leucine, glutamine and glucose
uptake into heartworm segments.

61
The last macromolecule that was looked at far
permeability was the drug ivermectin. Ivermectin is a potent
anthelmintic that has been used for heartworm prevention at
low doses. At these doses the drug does not have an effect
on the adult heartworm. Ivermectin has also been used to
control roundworm in several species. It 'was therefore

postulated that the difference might be in its ability to
enter the parasite. Both Ascaris suum and D. immitis segments were

perfused with APF while incubating in me with radioactive
ivermectin. The perfusion was carried out over a one hour
period per segment. Following the perfusion the perfusion
pump was turned off and the segment left in place for 20
minutes to allow for the possible accumulation of ivermectin
in the segments. The results of these experiments are shown

in Figure 16.

DPM/min/cm2 Surface Area

62

_N Cu
O b
O O
O O
-A:.. ..:..

O
O
O
111‘.
I

 

 

Heartworm 4 Ascarid

Figure 16: The permeability of both Ascaris suum and Dirofilaria
immitis to the drug ivermectin.

63

Figure 16 shows the results of the ivermectin

experiments. As can been seen the bars for both Ascaris suum

and Dirofilaria immitis are missing this is because in neither

case did any ivermectin enter the segments. When the segments
were allowed to "soak" in the incubation bath for twenty
minutes still no ivermectin entered either segment.

The next series of experiments involved the permeability
of both the heartworm and ascaris to different ions. The ions

looked at were 22Na“, 36Cl', and “Ca”

. 4uCi of radioactive ion
was added to 1m“ and the segments were perfused with APF.
The results for these experiments are shown in figure 17 and
table 4. As can be seen there is quite a large difference
to the permeability to the ions between the two parasite. The

ascaris seems to remain quite impermeable to ions as compared

to heartworm.

64

 

D. immitis A. suum
c1' 2085 i 196 28 i- 8
Na“ 795 i 100 9 i 2
Ca” 544 i 191 42 i 17

Table 4: The permeability of both Dirofilaria immitis and Ascaris
suum to ions expressed in DPM's.

65

 

 

 

 

 

 

 

 

w l“—
N 1
LE) 1
\ 113—6--
E
E
> 1E—7-~
E
_L
1E—8 -__ 1:10. immitis
35 -A. suum
l: j
1E—9 T a
Ca++ Na+

 

Figure 17: The permeability of both Dirofilaria immitis and Ascaris
suum to ions.

66

The ascaris tegument was inverted and perfused, as
described in the methods section. In this preparation APF was
on the outside of the tegument which represents the inside
of the parasite. In", was on the inside of the preparation
being perfused through the system but this represents the
outside of the parasite. The radioactivity was placed on the
outside of the system in the APF and the perfusate was
measured for the presence of the radioactive ions. Figure 18

and Table 5 shows the results from these experiments.

67

 

Into Out-of
NA” 9:2 18:4
c1' 28:8 53:6

Table 5: Net movement of ions into and out-of Ascaris suum
expressed as DPM's.

60-

50"

40‘

30*

20-

10‘

 

DP M/min/cm

68

 

 

 

Chloride

 

Sodium

 

 

 

O t-Of

)n-TO

Figure 18: Net movement of ions into and out-of Ascaris qum
expressed in Molar concentrations.

69

The movement of Na+ into the tegument of the ascarid was
not significantly different from the movement of sodium out-
of the preparation. In either' case the :measurement of
radioactive sodium was barely larger than control radiation.
Therefore the actual numbers might contain a higher degree
of error than if they were much larger than control.

Chloride movement out-of the tegument was significantly
larger than the chloride movement into the tegument. This
movement also occurred against a concentration gradient,

however the electrochemical gradient was not known.

70

Conclusions and Discussions

The tegument of both parasites remained intact and
undamaged throughout these experiments. This was demonstrated
by the fact that inulin was not able to enter the tegument
of either parasite.

Water was able, however, to enter both parasites. This
movement of water into the heartworm segment was
significantly greater than that into the ascaris segment.

Looking back at table 2, heartworm was over 18 times more
permeable to water than the ascaris. This difference could be

explained by the difference in the thicknesses of these two
parasites. However, this difference in thickness could not
explain the other differences seen throughout these
experiments

The first nutrient that was looked at was glucose. As
was seen in table 2, glucose was much more (approximately 30
times) permeable in the heartworm than the ascaris segments.
The glucose uptake in the heartworm segments was sensitive
to temperature. When the temperature was decreased from 37%:
to 49C there was a significant decrease in glucose uptake
which was reversible when the temperature was returned to
37°C. Figure 11 showed a temperature response curve for
glucose uptake in heartworm segments. However the TN“ could

not be calculated since the uptake of glucose was still

71
increasing at a temperature at which tegument began to

breakdown. This temperature dependency did not occur in
Ascaris suum where no decrease in glucose uptake occurred as a

result of lowering temperature.

APF contains glucose at a concentration of 10mM. When
the glucose was removed from the APF in the Same experiments
there was a significant decrease in glucose uptake in the
heartworm segments. Removing the glucose from the APF, made
the inside of the parasite devoid of glucose and actually
increased the glucose gradient. This fact signifies that the
uptake of glucose is sensitive to internal glucose and may,
in fact, depend upon it. This again didn't occur in ascaris

segments.
Glucose uptake into Dirofilaria immitis was also concentration

dependent. Glucose at concentrations between 0 and 11.1 mM,
in law, was examined for its effects on glucose uptake.
Since the amount of "hot" or radioactive glucose remained
constant in the incubation bath and the cold glucose could
displace it from being taken up. This signifies a saturable
mechanism. Figure 14 showed the curve that.was calculated for
this series of experiments. The actual experimental uptake
could be broken down into a diffusional part, that was linear
with respect to concentration; and a presumably mediated

process, that was saturable. From this mediated part of

72
uptake apparent enzymo-kinetic parameters could be
calculated, and was shown in table 3.

Thiacetarsamide, which is an arsenical used for the
treatment of heartworm infection in dogs, had a significant
but reversible affect on glucose uptake. The fact that this
inhibition was reversible might explain the necessity of
treating infected dogs for 3 consecutive days with this
compound to ensure complete efficacy in killing the adult
heartworms. The mechanism of action of this arsenical is
believed to be in its ability to inhibit sulfhydryl
containing enzymes (Booth, Veterinary Pharmacology and
Therapeutics).

Trans-tegumental glucose uptake into heartworm segments
is now believed (by this author) to be a process mediated by
a transport protein because the trans-tegumental uptake of
glucose into heartworm segments:

1) is temperature sensitive.

2) is concentration sensitive

3) is stereospecific for d-glucose

4) is sensitive to internal glucose concentration.

5) is sensitive to external but not internal pH.

6) is sensitive to the sulfhydryl inhibitor
thiacetarsamide.

7) is not sensitive to classical glucose transport
inhibitors applied to the outside of the
tegument.

8) is not coupled to an external sodium transport.

73

Flemming (1984) reported that ascarides might be more
permeable to macromolecules than previously reported because
they were able to measure glucose entering their preparation.
However the amount entering was small and corresponds nicely
with the values that I obtained in these experiments. Unlike
heartworm segments this amount was small and was not
sensitive to temperature or concentration, and therefore is
probably due to a simple diffusional process.

Acetate has received a lot of interest lately because it
is now believed that ascaris is capable of not only excreting
acetate but also, under glucose deprivation conditions, they
are able to utilize it as an energy source. Ascaris was not
able to transport any acetate into their body but heartworm
showed a very active mechanism for trans-tegumental uptake.
This uptake:

1) was temperature sensitive

2) was concentration sensitive, in that excess
cold acetate could displace the hot acetate.

3) also occurred against a large concentration
gradient of over 125 fold.

With regard to amino acid uptake by both these
parasites, we examined the transport of several amino acids,
representing the various "R" groups. The amino acids used
were leucine (which is non-polar and uncharged), tryptophan
(which is also non-polar and uncharged but samewhat larger),

asparagine (which is polar but uncharged), glutamine (which

74
is polar and uncharged, glutamic acid (which is polar and
negatively charged), and arginine (which is polar and
positively charged).

In the ascaris segments none of these amino acids were
able to enter through the tegument. In.the heartworm.segments
all of amino acids were able to enter through the tegument.
They all showed both concentration and temperature
sensitivity except for tryptophan. For leucine and glutamine
actual kinetic values (me and K“) could be calculated for
their uptake. Tryptophan on the other hand didn't show any
concentration sensitivity in the range tested. The graph of
uptake verses concentration was linear, signifying this was
a diffusional mechanism.

In heartworm segments the uptake of most amino acids
tested appeared to be a controlled process because:

1) It is temperature sensitive

2) It concentration sensitive, in as much as

excess cold nutrient could displace the uptake
of the labelled nutrient

Although the results are not shown several experiments
were performed measuring the accumulation of glucose,
acetate, and various amino acids by ascaris segments in order
to try to maximize the experimental conditions for uptake.
There are several recent reports of different media's being
used for ascaris incubation so it was decided to try several

of these in our system. We examined 1) APF on both sides of

75
the segment, 2) Ian" on the inside and APF on the outside and
3) Donahue's on the outside with APF on the inside. All of
these configurations provided the same results with regard
to trans-tegumental uptake.

Also, time has been reported to be a possible factor in
that a delay might exist between the start of the experiment
and the start of trans-tegumental uptake. Therefore, the
segments were also perfused for up to one hour. Increased
perfusion time didn't change the amount or rate of labelled
material passing through the parasite's tegument.

One experiment that needs to be done is to try a new APF
media reported to show interesting affects on ascaris muscle
preparations. This media was reported by Holden-Dye (1988)
with the main difference between in and Donahue's media,
being a high concentration of Mg++ and a slightly higher pH
(pH=7.6). Although I suspect that this new media will not
affect the results significantly, the experiment still needs
to be done.

Ivermectin is a rather interesting drug. It is used in
low concentrations in dogs for heartworm prevention. At these
concentrations the drug has no affect on the adult heartworm
but is a powerful microfilariacide. However at higher doses
ivermectin is known to have a detrimental affect on

spermatogenesis and oogenesis. Ivermectin is also used for

76
the control (or treatment) of roundworm (ascaris) infections
in many species.

The reason for this difference in its efficacy for the
treatment of heartworm and ascaris infection is not known.
One theory is that there is a difference in the parasites
ability to take up this drug and thus allow it access to its
site of action, which at this point in time is believed to
be its pharynx. Ivermectin's ability to penetrate these
parasites was tested and I observed no trans-tegumental
transport. A theory for this difference is that if
ivermectin's site of action is the pharynx, access to it in
the case of ascaris might be by oral ingestion. While in
heartworm if the parasite takes up all its nutrients trans-
tegumentally and oral uptake does not normally occur then
the access to the pharynx is therefore limited

The last group of experiments that was done was to look
at the permeability of ions in our preparations. Sodium
(ZZNAU), chloride (3°Cl'), and calcium (“Ca”) were used. In
all three cases the heartworm segments were much. more
permeable to ions than was the ascaris segments. Heartworm
was approximately 100 times more permeable to Cl', 800 times
more permeable to naf, and 10 times more permeable to Ca++
than ascaris, which appeared to remain impermeable to calcium

and sodium.

77

In ascaris the amount of radioactive sodium and calcium
was barely above background radiation. Chloride permeability
was higher than sodium and calcium. In the last series of
experiments ascaris segments were inverted and perfused as
described in the results section and the [permeability to
sodium and chloride was determined. Sodium movement in either
direction (both into and out-of) the segment was
statistically the same. While chloride movement was
significantly higher in an outward direction as compared to
an inward direction. In either direction this movement was
barely above control. This net movement of chloride outward
occurred against a concentration gradient. Although the
electrochemical gradient was and is still not known. It has
been reported (Wright 1987) that chloride concentration in
the pseudocoel cavity is lower than expected simply from the
concentration of positive ions. It has also been reported
that chloride might.play an important role in the maintenance
of this internal turgid pressure and from this data that

possibility seems quite feasible

 

78
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