Cuticular Amino Acids in Dirofilaria Immitis

Loyola University Chicago Loyola eCommons

Master's Theses Theses and Dissertations

1975

Bonnie Anne Ozuk Loyola University Chicago

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Recommended Citation Ozuk, Bonnie Anne, "Cuticular Amino Acids in Dirofilaria Immitis" (1975). Master's Theses. 2816. https://ecommons.luc.edu/luc_theses/2816

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:Bonnie Anne Ozu.k

A Thesis Submitted to the Faculty of the Graduate School of

Loyola University of Chicago in Partial Fulfillment of the Requirements for the Degree of

Ml.ster of Science

February .> \ \ ..... 1975 ACKNOWLEDGJill•IDi"TS

I would like to thank my advisor, Dr. Benedict Jaskoski, for information and guidance given me during the preplra.tion of this thesis. I also wish to thank Dr. Edward Pa.linscar and Dr. A. s. Dhaliwal for their help and ·work as committee members.

Dirofilaria immitis specimens used in this investigation were acquired from Dr. George Tulloch of San Antonio, Texas. I would also like to thank the Loyola biology faculty and fellow graduate students for their interest in the project. Special thanks to Mary Brokans for her support and encourag-ement, my family ' for their understanding, and Raymond :Moloney for the photography.

ii VITA

:Bonnie Anne Ozuk, daughter of Charles Ozuk and Georgian (Heaney) Ozuk, was born on September 11, 1950 •

. After graduation from Saint Cornelius grammar school in 1964, she attended The Imma.culata high school from 1964-1968. She re ceived a B:!.chelor's Degree in Biology in 1972 from Loyola University.

At Loyola, she became a member of Tri Beta, a biology honor society, and Circumference, a women's honor society. Remaining at Loyola to pursue a master's degree, she was a graduate teaching assistant for the next four semesters in micro biology. In 1974 she joined, Alpha Sigma Nu, the Jesuit honor soci ety. She received the Master of Science Degree in Biology in Feb ruary, 1975.

iii TABLE OF cmm:NTS

!age

ACKNOWLED@1El~S •••••••••••••••••••••••••••••••••••••••••••••• ii

VITA•••••••••••••••••••••••••••••••••••••••••••••••••••••••••• iii

LIST OF TABLES ••••••••••••••••••••••••••· •••••••••••••••••••••• iv

LIST OF FIGURES••••••••••••••••••••••••••••••••••••••••••••••• v

~'"TRODUCTION •••••••••••••••••••••••••••••••••••••••••••••••••• 1

LITEilATURE REVIEW•••••••••••••••••••••••••••••••••••••••••••••

Life Cycle ••••••••••••••••••••••••••••••••••••••••••••••••• 3 The Cuticle ••••••• ~·••••••••••••••••••••••••••••••••••••••• 6 Distribution in Illinois ••••••••••••••••••••••••••••••••••• 9 Significance of Dirofilariasis as a Zoonosis••••••••••••••• 11

11A.TER.IALS AND METHODS ••••••••••••••••••••••••••••• •, •••••••••••

Cuticle Removal •••••••••••••• ~ ••••••••••••••••••••••••••••• 13 Lipid Extraction••••••••••••••••••••••••••~•••••••••••••••• 13 Acid Hydrolysis •••••••••••••••••••••••••••••••••••••••••••• 14 Thin Layer Chromatography •••••••••••••••••••••••••••••••••• 15 Semi-~uantitative Measurements••••••••••••••••••••••••••••• 16 Veterinary Survey•••••••••••••••••••••••••••••••••••••••••• 16

RESULTS••••••••••••••••••••••••••••••••••••••••••••••••••••••• 18 \ Amino Acid Survey•••••••••••••••••••••••••••••••••••••••••• 18 S.em.i-Quanti tati ve Measurements •••••••••••••••••••••••••••• • 18 Veterinary Survey Results•••••••••••••••••••••••••••••••••• 18 DISCUSSION •••••••••••••••••••••••••••••••••••••••••••••••••••• 22

S'Ulvn·1A.R.Y • • • • • • • • • • • • • • • • • • .,. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 29 Bll3LIOGRAPHY •••••••••••••••••••••••••••••••••••••••••••••••••• 30 LIST OF TABLES

Table Page 1. Ri' Values of Amino Acids in Adult Female D1rofjlarja immjtjs Cuticle•••••••••••••••••••••••••••• 20 2. Am:ino Acids Identified in Nematode Cuticle••••••••••••• 26

iv LIST OF FIGURES

Figure Pc:l.g-e

1. Preserved }13.le and Female Dirofilaria immitis Specimens...... 4 2. Two Dimensional Chroma.togTalll of Acid Hydrolysate...... 19 3. Dog Heart with J.hssive Dirofilaria immitis Infection.... 24

v INTRODUCTION

Dirofilaria immitis (Leidy, 1856), the dog heartworm, is a nematode parasite transmitted to the definitive host by the mosquito. The definitive host for this :parasite is primarily the dog but other carnivores such as the cat and fox may also become in- f ected.

Taylor (1960) found that the nematode develops for seventeen days within the H3.lpighian tubules of the mosquito after being in- gested in a blood meal. After transmission to the dog, the heartworm continues development for three months in intermediate locations within the host according to Kume and Itagaki (1955) and Orihel (1961.) The parasite may continue its development in submuscular tissues, subcutaneous tissues, the subserosa of the veins, or in adipose tissues. According to Taylor (1960) the larva molts twice in the mosquito and Orihel (1961) describes the final two molts which occur in the dog. After the final molt, the heartworm migrates \ to the right side of the heart and pulmonary artery where it undergoes final maturation for an additional three months.

Anthelmintics necessary to control the spread of this parasite, which recently has spread inward from the eastern and southern seacoast regions, must penetrate the cuticle, an acellular layer secreted by the hypodermis. Fa.irbairn (1957) demonstrated that the cuticle or outer barrier of the worm is composed primarily of protein

1 2

with ~races of lipid and carbohydrate. Savel (1955,) :Bird (1956, 1957,) and Watson and Silvester (1959) performed amino acid analyses

on the Ascaris lumbricoides var ~ cuticle and Viglierchio .and Gortz (1971) performed a similar study on Anisakis physteris, an ascarid found in the sperm whale. :Bird (1954, 1958) also determined the amino acids present in the larval sheaths of Baemonchus contortus,

Trichostrongylus spp., and Nipuostrong;ylus muris; plus the amino acids found in the cuticle of the adult female Meloidogyne nematode, a

plant parasite. :Because anthelmintics must penetrate the cuticle to

be effective, knowledge of t.~e amino acid content of the cuticle which

is primarily protein is important.

The intent of the present investigation is

1. · the deterr~ination of the amino acid content of an acid hy drolysate of adult female Dirofilaria inunitis cuticle using thin layer chromatography

2. the performance of a semi-quantitative study of the amino acids found in the cuticle · 3. a survey of practicing veterinarians in six northern Illinois counties to discover the prevalence of dog heartworm in this area

------·--- -~·-- - -- LITERATUHE ?..EVIEW

Life Cycle Dirofilaria imrnitis (Leidy, 1856), the dog heartworm, is in the phylum Aschelminthes, class Nematoda. The spindle shaped male has a blunt spirally coiled posterior end and measures 120-200 mm in length; the female is rounded posteriorly and measures 250-310 mm. (Figure 1)

1"".anson (1878) first showed that a filariid worm, Wucheria ~ crofti, could infect the mosquito, a bloodsucking arthropod. The mos quitoes Cu.lex guinguefasciatus and £. pipiens have been identified as intermediate hosts for this nematode :i;arasite. Grassi and Noe (1900) working with ll• immitis determined that this :i;arasite also develops within the mosquito. Bancroft (1903 as cited in Kume and Itagaki 1955) l!}3.ve evidence that 12,. immitis is transmitted to the final host by the mosquito during its blood meal. Using phase contrast microscopy, Taylor (1960) traced the de velopment of the dog heartworm in the mosquito, Aedes aep;ypti, and found that the developmental cycle in the 'mosquito took seventeen days. After ingestion in a blood meal, the first stage larvae remain in the gut of the mosquito for twenty four hours before migrating to the Ma.lpighian tubule cells. Remaining for seven days in these cells, the larvae grow and become sausage shaped. ·On the seventh day, the larvae leave the cells and continue their development in the lumen of the Malpighian tubule cells.

3 '4

Figure 1 Preserved Ihle and Female Dirofil a ria immi tis specimens

-- 5

·During development in the mosquito, the heartworm larva molts

twice. According to Taylor (1960), the first molt occurs within the

tubule cell lumen on the tenth day. The second molt also occurring in

the cell lumen is on the thirteenth day. Completing its development in the cell lumen, the third stage or infective larva then migrates

to the mosquito's proboscis. At this point, the heartworm larva is ready to be transmitted to the final host. Other mosquitoes which

serve as the intermediate host for the heartworm are Aedes sollicitans, A. taeniorhynchus, Culex pidens, and Q.. guinguefasciatus. Kume and Itagaki (1955) outlined the passage of the infective larva once it enters the dog until it takes up permanent residence within the right side of the heart or pulmonary artery. After enter

ing percutaneously, the larvae migrate directly through the tissues

to submuscular tissue, subcutaneous tissue, subserosa of the veins, or adipose tissue. Final migration to the heart occurs through the venous system. Orihel (1961) observed larval development in the dog as the larvae migrate to the heart. The third stage larva molts by the twelfth day in the dog with the resulting fourth stage larva develop

ing until the seventieth day. After the final fourth molt which then occurs, the adolescent worm migrates to the heart and the pulmonary artery wherein the worm undergoes final maturation. According to Orihel (1961), the prep3.tent of canine dirofilariasis is 6.5 months, which is slightly lower than that described by other investigators.

Be noted that the difference was probably due to the number of worms r 6

in the initial infection. Newton and Wright (1956) discovered a dog filariid other than

~. immitis in the United States. Circulating microfilaria in the

dog's bloodstream no longer automatically indicates dog heartworm. The seccnd dog filariid, identified as Dipetalonema reconditum, is a

harmless inrasite found in the dog's subcutaneous tissues. The possi bility exists that earlier Dipetalonema. infections were mistakenly iden tified as heartworm infections. Newton and Wright (1956) support this contention. Their investigation showed that the dog heartworm develops

only in the mos~uito and Dipetalonema develops in fleas in the genus

Ctenocephalides. Prior to this investigation, D. immitis was also

thought to develop in the flea.

Control of the heartworm depends upon an effective anthelmintic

which penetrates the cuticle or outer covering of the worm. This a

cellular layer which protects the worm from the enviroilffient and nema.to

cides is secreted by the cellular hypoderrnis which underlies it. \

~Cuticle

Investigators agree that besi~es \later, the nematode cuticle is

composed pri.m:3.rily of protein along with small amounts of lipid and carbohydrate. Chitwood (1938) demonstrated a lipid film on the cuti

cle surface of Ditylenchus and Rhabditis. Trim (1949) and Bird (1957) also found such a film present on the cuticle of Ascaris lumbricoides

var~· Bird (1957) also found a carbohydrate in association with the Ascaris cuticular protein. Following acid hydrolysis of the cuti

cle, his investigations demonstrated an aldohexose. 7

. There is general agreement that the nematode cuticle consists of three basic layers: the outer or cortex layer, the middle layer, and the innermost or basal layer.

The protein found in the inner two layers is closely allied to collagen in its physical and chemical properties and is generally re- f erred to as collagen. Much controversy centers around the nature of the protein in the cortex layer. The sulfur content of this layer ex amined by Chitwood (1936) and Flury (1912) plus the ratio of histidine lysine-arginine found in the cortex by Savel (1955) show the cortex protein to be keratin. However, the X-ray diffraction of the cortex performed by Faure-Fremiet and Garrault (1944), and the solubility of the cortex in hot dilute alkali demonstrated by Fairbairn (1957) rep resent p:i.rt of the evidence sho~ing this protein to be collagen. Bird (1969) suggesting that it is inaccurate to comp:i.re proteins of invertebrates and vertebrates wrote that perhaps ''nematode cuticles are made up of secreted collagens unique to and characteristic of the Nematoda ••• ". \ Several studies have dealt with the amino acid content of the entire cuticle of the adult nematode plus the sheaths of various larval stages. For example, Bird (1954) identified nine amino acids in the bydrolysate of mixed larval sheaths of Haemonchus contortus, Tricho strongylus fil?.£•, and Nippostrong;ylus muris. He also worked with pure samples of Haemonchus contortus. The third stage larval sheaths of these nematodes contained the amino acidss proline, bydro:xyproline, aspartic acid, cysteic acid, glutamic acid, alanine, leucine, glycine, and valine. 8

. Savel (1955) working with the cuticle of Ascaris lumbricoides

va.r ~ identified thirteen amino acids in the whole cuticle. Using paper chromatography, Bird (1956) isolated the following amino acids in whole cuticle hydrolysates of Ascaris, Toxocara, and

Strongylus: glycine, alanine, valine, leucine, threonine, cystine,

cysteic acid, aspartic acid, glutamic acid, lysine, arginine, histi-

dine, tyrosine, tryptophan, proline, and hydroxYproline. Bird (1957) also surveyed the amino acids present in each cuticular layer of Ascaris. For this particular investigation, the iso-

lation of the amino acids was performed by Rees and Baston (unpublished

data) using column chromatography. They identified aspartic acid,

threonine, cystine, serine, proline, glutamic acid, glycine, alanine,

valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, and arginine. Rees and Baston (unpublished data) did not isolate the amino acid hydro:xyproline although it had been demon strated in Bird's earlier investigation (1956.) Watson and Silvester (1959) analyzed the whole cuticle of Ascaris \ as well as the collagen fraction of the cuticle. Comparison of their

results of the whole cuticle with those achieved by Bird (1957) shows

general agreement in the quantities of amino acids present. One ex- ception is the amino acid valine. Watson and Silvester (1959) found much less of this amino acid than Bird (1957.) Bird (1958) identified sixteen amino acids in the cuticle of a

plant p:i.rasitic nematode in the genus Meloidogyne. In a prepared

hydrolysate of adult female cuticle, he identified the amino acids:

- 9

glycine, alanine, threonine, cystine, cysteic acid, valine, leucine,

isoleucine, aspartic acid, glutamic acid, arginine, tyrosine, lysine,

histidine, proline, and hydroxyproline. Viglierchio and Gortz (1972) investigated the amino acids in the cuticle of Anisakis ;physteris, an ascarid found in the sperm whale, as

well as in the reproductive organs and the perienteric fluid. Cuticle analysis was done using a single column, high resolution automatic ion

exchange procedure. Proline, hydro:xyproline, aspartic acid, glutamic acid, alanine, leucine, glycine, valine, threonine, cystine, lysine,

arginine, histidine, tyrosine, tryptophan, serine, methionine, isoleu

cine, phenylalanine, and ornithine were identified in the cuticle. Re sults also indicated that 69 percent of the cuticular amino acid nitro

gen was contained in glutamic acid, glycine, serine, proline, and ar

ginine. Some differences were readily apparent when these results were comrared with Bird's investigation (1957.) For example, the cuticle of Anisakis contained a larger amount of phenylalanine than the Ascaris

cuticle. However, both cuticle samples contained large quantities of

proline, glycine, and arginine.

Distribution in Illinois

Once confined to eastern and southern seacoast areas, Dirofilaria immitis has been found in other regions of .the United States such as

the Midwest. Few studies have been done in Illinois concerning the distribution of this parasite. McKinney (1962) found that 1.4 percent of the two hundred twelve dogs he examined in Champaign county, Illinois were infected with D. immitis. In another investigation, Marquardt and

------~------~-~------~- 10

labia.rt (1966) indicated that 10.4 percent of the dogs examined in northenl Illinois counties were infected; 21.9 percent in the central counties harbored the para.site; and 32.6 percent of the dogs examined in the southern counties had the infection. A concurrent investigation

of Dipetalonema. incidence in Illinois by these two men revealed a 2.5 percent infection percentage in northenl Illinois counties, a 16.4 infection rate in the central counties studied, plus a 6.3 percent in fection percentage in the dogs examined in the southern Illinois coun

ties. The Illinois State Veterinary Medical Association's survey (1972 as cited in Illinois State Veterinary Medical Association News Release-- 1974) showed 5,619 positive cases of D. immitis infection in 45,000 dogs tested, or a 10.3 infection percentage. Their results agreed with those of Marquardt and Fabian (1966) which showed the highest incidence of 1!• immitis infection in Illinois is located in the southenl section of the state.

In northern Illinois, a heartworm s~ey undertaken by Flynn (1972) in Lake county revealed the highest infection rate in examined dogs was in January; the lowest infection percentage for the year re ported in September. In addition, a survey of veterinarians in six northern Illinois counties conducted by the author of this pa.per {1973 unpublished data) shows that although the heartworm is not an over whelming problem in this region, its effect is not to be overlooked by p:racticing veterinarians or by pet owners themselves. There is an in creasing awareness of ~. irmnitis in Illinois plus a concern for its effects in the dog. Perhaps this parasite is indigenous in Illinois 11 and has been demonstrated only now during this period of increased awareness and concern.

Illinois is not the only Midwestern state which must deal with the dog heartworm. A recent survey by Graham (1974) showed the infec tion percentage of~. irnmitis in northeastern Kansas in 1972 to be 16.7 percent.

Significance of Dirofilariasis ~§!. Zoonosis While man is not the definitive host for dirofilarial worms, he can become infected with them. A review of dirofilariasis in the United States by Beaver and Orihel (1965) showed that the first case was reported in Florida in 1.934. Al though incorrectly identified at the time, the worm was later shown to be a dirofilarial worm. Report- ing on thirty nine ca:::ies, Beaver and Orihel (1965) classified the in fections as 1) dirofilaria.l infections confined to the subcutaneous tissues, 2) worms other than dirofilarial worms located in subcutane ous tissues, and 3) dirofilarial worms present in the heart or lungs. \ These investigators determined that twenty five of the thirty nine cases were due to dirofilarial worms in subcutaneous tissues; three cases were ascribed to worms other than dirofilariids; eleven cases :in- volved worms located in the heart or lungs. They also observed that all the worms found and examined were sexually mature and were found only in adults, with women being infected more often than men. Exam- ination of the worms found in the heart and lungs indicated that these worms were indistinguishable from the dog heartworm, Dirofilaria immitis.

One of the non dirof ilariid worms examined was classified as a Dipetal- r 12

onema like species. The worms found in subcutaneous tissues are

thought to be Dirofilaria tenuis, which is normally parasitic in the

raccoon. Abadie, Swartzwelder, and Holman (1965) reported finding a non

gravid female !?_. immitis in a forty year old diabetic patient. Two other investigations by Jung and Espenan (1967) and Pacheco and Scho field (1968) have demonstrated microfilaria in the uterus of mature

female Dirofilaria tenuis .in subcutaneous locations in humans. A study

by Kahn and Meerovitch (1968) demonstrated a sexually mature worm in a

four year old child in eastem Canada. A recent report by de Carneri (1973) stated that one hundred cases of human dirofilariasis have been reported. These studies indicate that man is not totally unsuited for

dirofilarial worms normally found in animals.

\ r

MATERIALS AND METHODS

Cuticle Removal Cuticle was removed from eight thawed female Dirofilaria immitis pieces weighing a total of 87 mg. To remove the cuticle, the dehy drated worm segments were cut into one inch pieces and ground in a tissue grinder containing 6 ml triple distilled water. Grinding con tinued until the cuticle was noticeably loosened when viewed under the

dissecting microscope (45x.) A scalpel and probe were used to remove

any non cuticular material adhering to the cuticle. All work was done with the aid of the dissecting microscope. The pure cuticle sample was then washed three times in triple

distilled water in a centrifuge at 925 rpm for ten minutes. The su-

pernatant "1a.S removed with a disposable Pasteur pipette following each

wash and an additional 10 ml triple distilled "1ater was added. After the washings, the sample was dried for five days in a desiccator. The dried cuticle sample weighed 11 mg. \

Lipid Extraction

Cuticular lipid was separated from cuticular protein. The dried

weighed cuticle sample was placed in a 250 ml Erlenmeyer flask contain

ing 25 ml diethyl ether. To prevent oxidation of the flask contents,

the flask was covered with aluminum foil before placement on the Jru.rrell wrist action shaker. Six times during the next forty eight hours, the flask was removed from the shaker and the contents centri-

13 r 14

fuged ·at 3000 rpm for four minutes. The supernatant containing the

lipid fraction was decanted and stored in the refrigerator for lipid

analysis at a later date. The cuticle was resuspended in an additional

25 ml diethyl ether and then returned to the shaker after each centri- :fugation.

~ Hydrolysis

Following lipid extraction, the cuticle sample was placed in a

50 ml beaker containing 3 ml fresh diethyl ether. The excess was removed with a disposable Pasteur pipette and the sample was evaporated to dryness at room temperature for ten minutes.

Four ml 6N HCl was then added to the beaker. The cuticle decom- posed into finer pieces with the addition of the acid. The beaker con-

tents were then poured into a 5 ml ampoule which was then sealed. Dur-

ing these prep:i.ra tions, a 600 ml beaker containing 300 ml mineral oil

was placed in a heating mantle. The temperature of the oil was calibrated at 110 c. Once the temperature was calibrated, the ampoule was

·, suspended by a wire attached to a ring stand for twenty four hours in the oil. It was then removed and the contents filtered. The result-

ing filtrate or acid hydrolysate was then evaporated six times to re-

move a:ny excess hydrochloric acid. Evaporations were performed on a watch glass placed four inches above a hot ·plate set at medium. For

each evaporation, 10 ml triple distilled water Wd.s added to the watch

glass. . After the last evaporation, the amino acid residue was dissolved

in 10 percent isopropa.nol and stored in the refrigerator in a screw cap 1_5

test tube. The pH of the hydrolysate was adjusted to neutrality with

the addition of .2M Gomori phosphate buffer.

!h.!!! Layer Chroma togra.phy Using the acid hydrolysate, one and two dimensional chromatogTaIIls

were run on Eastman Chroma.gram Sheets, Number 13181, with fluorescent indicator. The adsorbent was a 100 micron thick silica gel layer with a polyvinyl alcohol binder. Spotting was done with plain capillary tubes measuring 75 mm long with an inner diameter of 1.1-1.2 mm. Filled with the acid hydroly

sate, the capillary tube was touched to the chromatogram sheet sixty times; the amino acid standa;rds were spotted three times each. All spotting was done under a flow of cool air in order to keep the spots as small as possible and to aid in drying the spot. It was necessary

for each application of the hydrolysate or amino acid standard to dry completely before the next spot could be applied.

The solvent used for the one dimensional chromatograms and as the

first solvent in the two dimensional chromatograms was n-butanol:gla cial acetic acid:water (8:2:2.) The two dimensional chromatograms were dried at room temperature with cool air for five minutes before being rotated ninety degrees and placed in the second solvent, n-pro i:anol:water (7:3.) For both chromatogram types, the solvent advanced approximately fifteen centimeters on the silica gel sheets and the

time in each solvent was eieht to nine hours. After removal from the second solvent; the sheets were dried under

a cool air stream for five minutes and then sprayed with ninhydrin pre-

~ ~~~~~~~~------.-...------~------16 p:i.red according to Stahl (1965.) The ninhydrin sprayed sheet was dried under a hot air stream until the colored spots appeared on the dried sheet. Rf values were determined for the amino acids appearing in the acid hydrolysate and compared with the Rf values of the amino acid standards as an aid in identifying the amino acids present in the acid hydrolysate. The calculated values were also compared to those found by other personnel who performed similar studies in this laboratory.

One and two dimensional amino acid standards were also run and processed for Rf value comparison with the hydrolysate as mentioned above.

Semi-Quantitative Measurements

Transmitted light from each colored spot appearing on the two dimensional chromatograms of the acid hydrolysate was measured on a

TLC Densitometer, Model 530. The scan line was lined up with each spot and light readings were taken for each 1 mm as the sheet was advanced manually. The resulting values were compared to determine relative \ quantities of each amino acid present in the acid hydrolysate.

Veterinary Survey

Letters were sent to one hundred ten veterinarians in northern

Illinois counties in the summer and fall of 1973. Na.mes of veterinarians were chosen at random from a list of Illinois veterinarians supplied. by the American Veterinary Medical Association. Each surveyed veterinarian was requested to inform this investigator of the dog heartworm incidence encountered in his practice. The results from this 17 survey were then added to those compiled by another investigator from this laboratory. This second survey represented a determination of the incidence of _!!. im.~itis infection in the north and northwestern sub urbs of Chicago achieved by visiting veterinarians in this area.

\ RESULTS

·Amino ~ Survey Fifteen amino acids were identified on chromatograms of an acid hydrolysate of adult female Dirofilaria i.mmitis cuticle (Figure 2.)

Rf values were calculated for the amino acids which appeared on four two dimensional chromatograms. The Rf values plus the position of the colored spots on each chromatogram were comi:ared with an unpublished study done in this laboratory on Ascaris cuticle and also with Jas koski and :Butler's (1971) study of the tick egg shell amino acids. A two dimensional chromatogram spotted with control amino acids was then run alongside a two dimensional chroma.togra.m of the acid.hydrolysate. The following table shows which amino acids are contained in the adult female dog heartworm cuticle (Table 1.)

Semi-Quantitative Measurements

A ~lodel 530 TLC Densitometer was used to scan the spots which appeared on the two dimensional chromatograms of the acid hydrolysate.

Results of the scan indicated that the amino acids serine/glycine, glutamic acid, praline, isoleucine/leucine, and arginine were present in greater concentration than the other amino acids contained in the hydrolysate.

Veterinary Survex Results

Results from twenty two veterinarians in Winnebago, Boone, Mc-

18 19

lleu/Leu 1/\')Phe \~/

Oal

I 1! -·(\l, N' .. : ...... co

.. 'd ·ri : 0 c1S . O· •r-1 i +> : Cl) i o: c1S ';J Lys ·ri (~)Asp 0 al ; r-1 • bD • .. c 'd

t...... -.---·--·~-··- n-propmol:water (7:3) __...,.

Figure 2 Two Dimensional Chromatogram of Acid Hydrolysate 20

Table 1 Rf Values of Amino Acids in Adult Female Dirofilaria immitis Cuticle

Amino Acid Rf value* (Solvent 1)a Rf value* (Solvent 2)b

alanine/threonine .396 .453 !arginine .114 .202

asptrtic acid .199 .310 cystine .074 .254 glutamic acid .320 .270 hydroxyproline .326 .434 isoleucine/leucine .671 .633 lysine .• 085 .151

phenylalanine .678 .679 proline .338 .390 serine/glycine .216 .456 valine .574 ' \ .522

*composite Rf value · a) n-butanol:glacial acetic acid:water (8:2:2) b) n-propanol:water (7:3) 21

Henry; Lake, Cook, and Kane counties in Illinois indicate the presence

of dog heartworm in this area. Although canine filariasis is not con sidered a major problem, the veterinarians have reported treating a num ber of cases. Reported incidence percentages range from .3 percent in the Skokie area to a 10 percent incidence reported from :B3.rrington. These results agree with those of an earlier investigation undertaken in this laboratory which revealed that 9.01 percent of 3,439 dogs examined in the north and northwestern suburbs of Chicago harbored D. immitis. It is noteworthy, however, that these figures represent examinations of dogs brought to veterinarians. Many of the dogs may have sho'-m. symptoms of canine filariasis before they were brought in for a check-up. If this were the case, the actual incidence of dog heartworm would be smaller than that reported by the veterinarians.

\ DISCUSSION

Canine filariasis is now recognized as a potential problem in

Illinois. Results from surveyed veterinarians (unpublished data) in northern Illinois indicate that the dog heartworm has been found in Winnebago, Boone, Cook, Kane, Lake, and McHenry counties in Illinois.

A possible explanation for the spread northward of the dog heart worm is the increased interstate travel of dogs used in hunting and in dog shows. Work done by Todd (as cited in the Illinois State Veteri nary ~~dical Association news release 1974) of the University of 11- linois College of Veter:inary Medicine on the epidemiology of canine filariasis indicates that the microfilaria are capable of adapting to the lower temperatures in northern regions. Todd has also discovered infective larva in the three common genera of mosquitoes found in

Illinois.

Once established in an area, it is difficult to eradicate this para.site. It is important to become aware of the heartworm problem since D. irmnitis can cause infections both in man and the dog. Ef fective control of the spread of the heartworm and its eradication from a:ny endemic region is therefore necessary. One eradication method is the destruction of the intermediate host, the mosquito. Without the intermediate host, the :inrasite could not develop pa.st the microfilaria stage. However, with present day methods it is impossible to completely

22 23 eradicate the mosquito population in a given area. For example, it is believed that "the mosquito crop will be larger in the Chicago area this year because of the heavy spring rains. These rains have provided a suitable environment for the development of mosquito eggs laid two to three years ago. While continued effor~s should be employed to check the mosquito, work along this line will not supply the total answer.

A second control method is to concentrate on the worm itself and the develoµnent of an effective anthelmintic which would destroy the rarasite without adversely affecting the host. Anthelmintics presently in use, while somewhat effective against the nematode, are dangerous to the dog's health.

The adult heartworm resides in the right side of the heart and pulmonary artery (Figure 3.) They may also appear in other areas, such as the vena cavae. Repeated infections cause an increase in the num ber of adult worms present. A large mass of heartwo:rms in the heart can restrict blood flow and cause death by heart failure. Living worms in the pulmonary artery cause endarteritis and dead worms in the same location pose danger as possible emboli. It has also been determined that the liver and kidneys can be affected by the dog heartworm's pres ence in the body.

Anthelmintics presently used according to Iacheco's (1972) syn opsis of lfume 1 s symposium remarks affect different stages in the heart worm life cycle. Orf'P.IliC compounds which may or may not contain heavy metals are used as microfilaricides. Dithiazinine Iodide and organo phosphates are examples of such compounds. Migrating worms in the dog 24

Figure 3 Dog Heart with Massive Dirofilaria immitis Infection 25 are checked with diethylcarbamazine administered daily throughout the mosquito season and for two months after the end of the season. Or- ganic arsenic compounds, such as Cap:i.rsolate Sodium, kill adult worms present in the dog. It is best to keep the dog inside during treat- ment in order to avoid reinfection with the heartworm.

Any anthelmintic must penetrate the outer cuticular barrier of the heartworm. Because the nematode cuticle is basically protein as described by Fairbairn (1957,) an amino acid analysis of the adult female dog heartworm cuticle was undertaken. The next table outlines the results of several investigations of the amino acid content of the cuticle of Ascaris and Anisakis, a plant :rarasite Meloidogyne, and a . filariid worm,_!!. immitis (Table 2.)

The amino acid content plus the relative quantities of the amino acids present in the entire ma.le and female dog heartworm was determined by a worker in this laboratory using procedures previously described in this report. He found that the adult female heartworm contained lysine, histidine, arginine, aspa.rtic acid, glycine, praline, hydro:xyproline, serine, glutar.iic acid, alanine, threonine, phenylalanine, leucine, isoleucine, valine, methionine, and tyrosine. Trypto- phan was found present in an alkaline hydrolysate. Cysteic acid, lysine, serine, histidine, as:r;a.rtic acid, arginine, glycine, praline, hydro:xyproline, alanine, threonine, gluta.mic acid, valine, phenylalanine, leucine, isoleucine, methionine, and tyrosine were identified in the adult male nematode. Densitometer readings indicated that the female contained more as:r;a.rtic acid than the male but that the male worm had 26

Table 2 Amino Acids Identified in Nematode Cuticle

Amino Bird Bird Watson & This La bViglier- Bird Present Acid !ls caris Ascaris Silvester Ascaris *chio & Meloido- Study layers Ascaris Gortz gyne ~. immitis .AnisakiE

3.lanine + + + + + + + 3.rginine + + + + + + + asi:artic + + + + + + acid - tcystine + + + + + + oysteic + + acid ~utamic + + + + + + acid glycine + + + + + + + nisti- + + + + + + d.ine riydroxy- + + + + + + proline isoleu- + + + + + + cine leucine + + + + + + + lysine + + + + + + + nethio- + + + + nine :>rni- +

thine \ tphenyl- + + + + + alanine 1Proline + + + + + + + serine + + + + + rthreo- + + + + + + + nine ltrypto- + + + phan !tyrosine + + + + + + raline + + + + +. + +

* unpublished data. 27 more serine than the female.

A study of the results in Table 2 shows a general similarity in the cuticular amino acids of Ascaris lumbricoides var ~' Anisakis Physteris, Meloidogyne, and Dirofilaria innnitis. This similarity is noteworthy considering the wide range of habitats of these nematodes.

Ascaris is an intestinal parasite in the pig; Anisakis is found in the gastric lumen or attached to the mucosa of the sperm whale; Meloidogyne is a plant inrasite; and D. immitis is a blood inrasite in the dog. Fifteen amino acids were identified on completed chromatograms of an acid hydrolysate of adult female 1!• immitis cuticle. Because an acid hydrolysate was preinred, no check was made for the amino acid tryptophan. This a.milio acid is destroyed during this hydrolysis pro cedure. As mentioned, no significant differences are evident in an amino acid comparison. Cysteic acid, histidine, tyrosine, tryptophan, and methionine have been found in various investigations of the Ascaris cuticle but not in the heartworm cuticle. Histidine, methionine, trypto pha.n, and tyrosine were found in the Anisakis cuticle but not in the heartworm cuticle. The Meloidogyne cuticle contains cysteic acid, his tidine, and tyrosine which are not found in the heartworm cuticle. On the other hand, ;Q,. immitis cuticle contains phenylalanine and serine which have not been identified in the Meloidogyne cuticle. These cuti cular amino acid differences could be caused by the differences in the worms.

An amino acid comparison between the acid hydrolysate of adult female D. immitis cuticle and an acid hydrolysate of the entire female 28

worm.shows a variation in the amino acids histidine, methionine, and

tyrosine. These amino acids are present in the whole worm but not in

the cuticle. On the other hand, cystine was found in the cuticle but

not in the hydrolysate of the entire worm. Further investigation is

indicated in order to explain these differences.

Light transmittance readings taken on the densitometer indicated

that serine/glycine, glutamic acid, proline, isoleucine/leucine, and

arginine are present in the highest concentrations of the fifteen amino

acids identified. This ~e;rees with the results achieved by Viglierchio

and Gortz (1972) with Anisakis. They determined that the majority of the cuticular amino acid nitrogen was contained in glutamic acid, gly

cine, serine, proline, and arginine. Three groups of two amino·acids each were incompletely separated

on the chromatograms because of similar Rf values. These three groups

were alanine/threonine, serine/glycine, and isoleucine/leucine.

A study of the adult male].. i.mmitis cuticle would be of interest to see if any major qualitative or quantitative differences exist be

tween the two sexes. If a significant difference is found, it would then be necessary to formulate a different anthelmintic for each sex•

....1i------~~'~"--~,--·--- SUMMARY

Thin layer chromatography of an 11 mg sample of adult female

Dirofilaria immitis cuticle revealed fifteen amino acids. The iden tified amino acids were alanine, arginine, aspartic acid, cystine, glu tamic acid, glycine, hydroxyproline, isoleucine, leucine, lysine, phenyl alanine, proline, serine, threonine, and valine. A semi-quantitative study of these amino acids showed serine/glycine, glutamic acid, pro line, isoleucine/leucine, and arginine as present in the greatest con centration. A survey of six northern Illinois counties indicated once again the presence of dog heartworm in Illinois. The effects of this p:!Xasite both on the dog and humans must be recognized and treated in order to prevent canine filariasis from becoming a more important problem.

29 rI BIBLIOGRAPHY

' Abadie, s., J. Swartzwelder, R. Holman. 1965. A human case of~ filaria L"l!r.litis infection. American Journal of Tropical Medicine and Hygiene 14:117-118.

:Beaver, P., T. Orihel. 1965. Human infection with filariae of animals in the United States. American Journal of Tropical Hedicine and Hygiene 14:1010-1029.

:Bird, A. 1954. The cuticle of nematode larvae. Nature 174:362. 1956. Chemical composition of the nematode cuticle. Observations ----- of the whole cuticle. Experimental Parasitology 5:350-358. 1957. Chemical composition of the nematode cuticle. Observations - on individual layers and extracts from these layers in Ascaris lu.mbricoidescuticle. Experimental Parasitology 6: 383-403. 1958. The adult female cuticle and egg sac of the genus Meloido- - gyne Goeldi, 1887. Nematologica 3:205-212.

and J. Bird. 1969. Skeletal structures and integument of Acantho- - cephala and Nematoda, pp. 253-288. In M. Florkin and B. Scheer (eds.) Chemical Zoology. Academic Press, New York. Carneri, de I. 1973. Subcutaneous dirofilariasis in man-not so rare. Transactions of the Royal Society of· Tropical Medicine and Hygiene 67:887. Chitwood, B. 1936. Observations on the chemical nature of the cuticle of Ascaris lumbricoides var ~. Proceedings of the Hel minthological Society of Washington, D.C. 3:39-49. _ 1938. Jffirther studies on nemic skeletoids and their significance in the chemical control of nemic pests. Proceedings of the Helminthological Society of Washington, D.c. 5:68-75. Fa.irbaim, D. 1957. The biochemistry of Ascaris. Experimental Para sitology 6:49-54.

Fa.ure-Fremiet, E., H. Ga.rrault. 1944. Proprietes physiques de l'ascaro collagene. Bulletin Biologique de la France et de la Belgique 78:206-214.

30 r 31 Flury:, F. 1912. Zur chemie and toxikologie der Ascaroidea. Archives Experimental Iathology in Fha.rmacology 67:275-392. Flynn, R. 1972. Heartwo:rm survey. Annual report-1972. Lake County, Illinois. Graham, J. 1974. Canine filariasis in northeastern Kansas. Journal of Iarasitology 60:322-326. Grassi, B., G. Noe. 1900. The propagation of the filariae of the blood exclusively by means of the puncture of peculiar mosquitoes. British Medical Journal 2079:1306.

Illinois State Veterinary Medical Association News Release~1974. Jaskoski, B. v. Butler. 1971. Rhinicephalus sanguineus: Amino acid composition of egg shell. Experimental Parasitology 30: 400-406. Jung, R., P. Espenan. 1967. A case of infection in man with dirofilaria. American Journal of Tropical Medicine and Hygiene 16:172-175.

Kahn, A., E. Meerovitch. 1968. Zoonotic filarial infection in a 4-year old child in eastern Canada. American J ourna.l of Tropical Medicine and Hygiene 17:730-732.

lfu.me, s., s. Itagaki. 1955. On the life-cycle of Dirofilaria immitis in the dog as the final host. British Veterinary Journal 3:16-24. Mmson, P. 1878. On the developr.:ient of Filaria sanguinis hominis, and on the mosquito considered as a nurse. Journal of the Linnean Society of London (Zoology) 14:304-311. furqua.rdt, w., w. Fabian. 1966. The distribution in Illinois of filar iids of dogs. Journal of Iarasitology 52:318-322.

McKinney, R. 1962. The presence of D. immitis and Dip_etalonema §..£• microfilariae in dogs of Champa.ign County, Illinois. Illin ois Veterinarian 5:43-44. Newton, w., w. Wright. 1956. The occurrence of a dog filariid other than Dirofilaria immitis in the United States. Journal of Iarasitology 42:246-258.

Orihel, T. 1961. Morphology of the larval stages of~· immitis in the dog. Journal of Iarasitology 47:251-362. r 32

lacheco, G. 1972. Synopsis of Dr. Seiji Kume's reports at the First International Symposium on Canine Heartworm Disease, pp. 137-1A5. In R. Bradley (ed.) Canine Heartworra Disease- The Current Y.nowledge. Depa.rtment of Veterinary Science, Gainesville, Florida.

_____ and H. Schofield, Jr. 1968. Dirofilaria tenuis containing micro filaria in man. American Journal of Tropical Hedicine and Hygiene 17:180-182.

Savel, J. 1955. Etudes sur la constitution et la metabolisme protei ques d'Ascaris lUDbricoides Linne, 1758. Revue de Pathologie Generale et Comparee 55:52-121. Stahl, E. 1965. Th:in Layer Chromatography. Academic Press, New York.

Taylor, A. 1960. The development of Dirofilaria immitis in the mos quito, Aedes aegruti. Journal of Helminthology 34:27-38. Trim, A. 1949. The kinetics of the penetration of some representative anthelmintics and related compounds :into Ascaris lumbricoides var suis. Parasitology 39:281-290.

Viglierchio, D., J. Gertz. 1972. Anisakis physteris. Amino acids in body tissues. Experimental Parasitology 32:140-148. Watson, M., N. Silvester. 1959. Studies of .illvertebrate collagen preparations. Biochemistry Journal 71:579-586.

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APPROVAL SHEET

The thesis/atfof.fr/fffo/t/¥~/i submitted by Bonnie Anne Ozuk has been read and approved by members of the Department of Biology

The final COP.trs have been examined by the director of the thesis;J..ZJJJJ.J/JJJfUJ and the signiture which appears below verefies the fact that any necessary ~hanges have been incorporated and that the_thcsis/dJfa/11ffa/a/~IP/1 is now given final approval with reference to content and form.

The thesis/1/PNo/~fff/,?f is therefore accepted in partial fulfillment oi'the requirements for the degree of Master of Science in Biology .

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