• TABLE OF CONTENTS
HIDE
 Front Cover
 Introduction
 Literature review
 Acknowledgement
 Summary and Conclusions
 Bibliography






Group Title: Veterinary Science Mimeo Report
Title: The etiology of pathologic changes in canine heartworm disease
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 Material Information
Title: The etiology of pathologic changes in canine heartworm disease
Alternate Title: Veterinary Science Mimeo Report - University of Florida ; VY70-1
Physical Description: 20 leaves : ; 28 cm.
Language: English
Creator: Bradley, Richard E., 1927-
University of Florida -- Agricultural Experiment Station
Donor: unknown ( endowment )
Publisher: Florida Agricultural Experiment Stations
Place of Publication: Gainesville, Fla.
Publication Date: October, 1969
Copyright Date: 1969
 Subjects
Subject: Canine heartworm disease -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: Richard E. Bradley.
Bibliography: Includes bibliographical references (leaves 18-20).
General Note: Cover title.
General Note: "October, 1969."
General Note: "A portion of this paper was presented at the Fourth International Conference of the World Association for the Advancement of Veterinary Parasitology, Glasgow, Scotland, August 26-28, 1969."
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Resource Identifier: oclc - 433125234

Table of Contents
    Front Cover
        Page 1
    Introduction
        Page 2
    Literature review
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
    Acknowledgement
        Page 17
    Summary and Conclusions
        Page 18
    Bibliography
        Page 19
        Page 20
        Page 21
        Page 22
Full Text
OL)

v', 'o-'


VetArinary Science
Mimeo Report VY70-1
October, 1969


Flor'ida Agricultural
Experiment Stationj
Gainesville, Florila


TIL ETIOLOGY OF PATHOLOGIC

CHANGiS IN CANINE HifA TLORMi DISEASE







Richard E. Bradley, D.V.i., Ph.D.
Department of Veterinary Science
University of Florida
Gainesvillc, Florida 32601 U.S.A.








A Portion of This Paper 'as
Presented at the Fourth International
Conference of the Vorld Association
for the Advancement of Veterinary
Parasitology, Glasgow, Scotland,
August 26-2d, 1969


10/69:500












Introduction


Canine hea-rt.orm disease hae been recognized as a clinical entity

by veterinarians for many years. The current :ilnocledge of methods for

diagnosis and treatment of this disease was surmumarized in the proceed-

ings of a symposium held at the University of Georgia, Athens, Georgia,

in 1967. This information was recently published and is available for

reference (Jackson, R.F., et al., 1969).

The etiological agent of canine heartwoim disease is the nematode,

Dirofilaria immitis. The adult stage of this filarial worm inhabits the

heart and adjacent large blood vessels of the definitive host, the dog.

Although a considerable amount of information has been published

about certain aspects of the biology of the parasite and the clinical

signs associated with an infection of the adult parasites, very little

is known about the pathologic changes within the host during the prepa-

tent period. Similarly, little is known of the effects of a long-lived

adult infection and the microfilaremia that accompanies it.

This report is concerned with the causes of pathologic changes

that occur in naturally-infected dogs due to patent infection with

D. immitis and in non-infected dogs that were transfused with D. immitis

miciofilariae.








2-

Literature Review


I. Biology of the Parasite, Dirofilaria immitis.

The filarial worm, Dirofilaria immitis, was first described by

Gruby and Delfond in 1843 and named by Leidy in 1856. The life

cycle of this parasite was described by Fulleborn in 1908, who

shoved the requirement for a suitable mosquito intermediate host

for successful completion of the cycle. Other workers, starting

vith Breinl (1921), described developing filarial larvae from dog

and cat fleas (Ctenocephalides canis and C. felis, respectively),

thus giving support to the theory that several blood-sucking ar-

thropods were suitable vectors of D. immitis. This proposal of

multiple vectors received continued support until Newton and bright

(1956) described the presence of another filarial parasite of dogs

in the United States belonging to the genus Dipetalonema. The reve-

lation of existence of two filarial worms in dogs in the U.S. clari-

fied the confusion of multiple vectors, since dog and cat fleas are

apparently the vectors of Dipetalonema sp. and not of D. nimmitis.

However, restriction of D. immitis vectors to only mosquitoes

has not helped the confused state of incidence and distribution of

this parasite. Although some authors (Hu, 1931; Summers, 1943;

Kartman, 1953; Bemrick and Sandholm, 1966) state that various mos-

quitoes are suitable vectors of this parasite and give a list of

genera, species, and varieties, it has been our experience that

laboratory transmission of D. immitis is not readily accomplished.








-3-


Another relatively unknown feature of the biology of Dirofilaria

immitis is the incidence of the parasite and the epizootiology of

clinical heartworm disease, which is reported by practicing veteri-

narians to be quite high. The incidence of D. immitis may be meas-

ured by microscopic examination of blood samples and identification

of any microfilariae present from representatives of the dog popula-

tion in an area. These data may also be obtained at necropsy of a

sample of the dog population. However, by either method, valid data

a.e difficult to obtain and published information on the true epi-

zootiology of canine heartworm disease and the incidence of the para-

site is scanty. The recent report of Thrasher and Clanton (1968) rep-

resents an important first step in obtaining statistically valid and

scientifically sound data on incidence of D. immitis in dog popula-

tions in Georgia. Also in 1968, we conducted an incidence study in

Hlillsborough County (Tampa), Florida, utilizing the necropsy method

for determining incidence of the parasite. Although, admittedly,

precise history is lacking on the dogs, some interesting facts

were revealed; a summary of results of the survey iL presented in

Table I. The most striking feature of the study was the fact that

two-thirds of the dogs with D. immitis had less than 10 adult para-

sites. This finding was in contrast to the fact that the Hills-

borough County area has a heavy hosquito population vell-distribu-

ted among all species reported from Florida and the U.S. as suc-

cessful vectors of D. immitis. It should follow that the area is








-4-


enzootic for D. immitis infection and that native dogs would presumably

be exposed to a high degree of infection since mosquito populations are

present during the entire year. But the small numbers found in our

sample may indicate that an inhibitory mechanism is operating at some

stage in the life cycle which limits the number of adult D. immitis

present in a dog at any one time. Otto (Jackson, R.F., ct al. 1966)

and others have attributed this inhibition to an immune mechanism op-

erating in the dog host. Such may well be the case, but one cannot

overlook the indirect evidence that there are many factors that may be

exerted on the population of mosquito vectors in enzootic areas to

actually limit the exposure of dogs to infective larvae. Research a-

long this line has not been pursued with D. immitis. Attempts are cur-

rently being made in our laboratory to develop satisfactory serologic

methods of identification of filarial larval stages found in wild-

caught mosquitoes as a means of studying the infectivity rates of mos-

quitoes and thus the efficiency of transmission to the definitive host.

II. Pathologic changes during the prepatent period of D. immitis infection

in dogs.

In recent years, attention has been directed to possible changes

that occur in dogs infected with developing larvae of D. immitis. How-

ever, experimental data utilizing laboratory infected dogs is very limi-

ted.

The report of Kume and Itagaki (1955), which traced the course

of larval migration and development to completion, did not include data








-5-


on cellular changes along the migratory route. There have been addition-

al reports of experimental infections of dogs in the laboratory (Webber

and Hauking, 1955; Newton, 1957; Sawyer and Weinstein, 1963; neston, 1968),

but none have contained information on host response during prepatency

of the successful infections.

The only report dealing with changes in the host during the prepa-

tent period of experimentally-induced D. immitis infections has been that

of Pacheco (1966). He attempted to quantitate the immune response in

dogs that were inoculated with infective D. immitis larvae and reported

a rise in antibody detectable by indirect hemagglutination (IIA) by two

weeks after inoculation. Complement-fixing antibodies were detectable

at three months post-inoculation and the general pattern was a rise in

detectable antibody to a peak at approximately the time that patency of

the infection would occur. After that time, he reported that the titer

diminished to an undetectable level at about nine months post-inoculation.

However, there are several unanswered questions in Pacheco's report such

as prior and concomitant experience of his experimental dogs with other

helminth parasites and, perhaps the most important of all, confirmation

of completion of the D. immitis life cycle by either demonstration of

circulating microfilariae or necropsy data on the number of adult para-

sites that developed from the inoculations with infective D. immitis

larvae in his experimental doss.

A recent report that contained information on successful laboratory

infections of experimental dogs with D. immitis is by Tulloch et al.








-0-


(196J). The design of the experiment, however, was to evaluate a

program of preventive drug therapy and attention vas not devoted

to pathologic changes except to those seen at necropsy.

III. Pathologic changes during the course of patent infections with D.

immitis in dogs.

A sizeable number of reports on gross and microscopic lesions

attributed to the adults of D. immitis have been published. Compre-

hensive studies on pulmonary arterial lesions have been reported by

Adcock (1961), on hepatic lesions by von Lichtenberg et al. (1962),

and on general pathologic changes by Winter (1959) and others. All

infected subjects in these studies were naturally-infected and no

information of the length of the patent period, the number of infec-

tive larvae that the dogs had been exposed to, or the dynamics of the

parasite population was presented.

In the report by Tulloch et al. (196J), microscopic lesions in

the lung and liver tissues of the infected control (non-medicated)

dogs confirmed the descriptions of changes in these tissues by ear-

lier workers except for the appearance of an eosinophilic granuloma

in the kidney of one dog.

The effects of microfilaremia on infected dogs, either direct

or indirect, have not been extensively studied. There have been

suggestions of pathologic effects by several authors, but experimen-

tal proof has not been offered.








-7-


Uong (1964 a, b) reported on studies designed to determine the

mechanisms that control the level of microfilaiemia in dogs naturally-

infected with D. immitis and laboratory-infectod with Brugia pahangi

and the relationship to the immunologic responses of the host. How-

ever, although cellular or tissue responses to microfilaremia were

not reported, one of the significant contributions from these papers

was a technique for separating microfilariae from whole blood compo-

nents, following the method originally proposed by Yaegcr (1i60) for

trypanosomes.

A report that directly implicates microfilariae as being respon-

sible for "transfusion reactions" in dogs was made by Ota et al. (1962).

They reported that transfusing dogs with homologous blood containing

microfilariae produced a true anaphylaxis based on a filarial antigen-

antibody reaction. In a carefully-designed series of experiments, they

conclusively showed that the anaphylactic reaction occurred only in

cases where the transfused blood contained microfilariae. Similar re-

actions occurred in dogs injected intravenously with extracts of adult

D. immitis. The report did not include microscopic changes in tissues

after the reaction occurred, but the clinical signs and gross lesions

at necropsy were those generally accepted as due to anaphylactic shock

hypotensionn, increased portal venous pressure, and hepatic congestion

manifested as a swollen, firm, dark purple-colored liver at necropsy).

ImTARIALS AITD I;THODS

Experimental design. To study the effects of adult D. immitis,

naturally-infected dogs were necropsied and both gross and microscopic

lesions recorded.












In order to study the effects of microfilariaa and microfilaremia,

large quantities of microfilariae were separated from whole blood of

naturally-infected dogs and transfused into non-infected dogs held in

isolation from exposure to D. immitis infection. Clinical and necrop-

sy observations were made on the recipient dogs. Lesions were recor-

ded.

Natuially-infected dogs. iMixed breed dogs, obtained from a li-

censed animal dealer, and shown to have D. immitis microfilaremia by

microscopic examination of the blood were used. All animals were ex-

amined clinically and treated for intestinal helminths with appropri-

ate anthelmintic drugs, when diagnosed.

The dogs vere housed in individual outdoor, concrete-floored,

wire runs, roofed for weather protection. Feed consisted of free-
R
choice Gaines meal; drinking water was likewise free-choice.

Non-infected dogs. The principals in this phase of the study

were pedigreed Beagles obtained at weaning time from a commercial

colony maintained in an area in the United States known to be free

of D. immitis. This factor is monitored in all dogs in the colony

on a regular basis by microscopic examination of blood samples for

microfilariae. Fecal examinations for helminth ova are also done

on a regular basis and records show the colony to be essentially

helminth-free and totally D. immitis-free.

The experimental dogs were housed in Rockefeller-type isolation

rooms, double-screened, air conditioned, and entered only through a


- 8 -








- 9 -


double-door entry room. Protective clothing and rubber boots were

donned in this area prior to entering the isolation room; insecti-

cide spray and scrubbing with disinfectant was done as a routine to

prevent entry of mosquitoes into the animal quarters. All entry of

feed, utensils, instruments, etc., and removal of waste materials

was similarly under strict regulation.

The dogs were housed together as litters until the time of es-

trus of the females approached, at which time they were separated by

sexes. Feeding consisted of a commercially-manufactured dog food pel-
R
let (Gaines meal) which was "self-fed" from hanging feeders; drinking

water was provided ad libitum. All dogs were vaccinated against dis-

temper-hepatitis and rabies, at the proper times, using modified live

virus vaccines.

Preparation of microfilariae for transfusion. Heparinized whole

blood was collected under aseptic conditions from naturally-infected

dogs treated as outlined by Wong (1966 b). Slight modifications of

Long's method inclu:jd the collection of 300 ml. quantities of blood

and, after adding the phytohemagglutinin, the resulting mixture was

placed in conical, screw-capped, 26 mm. x 151 mm. glass centrifuge

tubes. The routine of washing and centrifugation (refrigerated cen-

trifuge, 1100 r.p.m. at 10 C.) was then completed. The total number

of microfilariae in the final sample was estimated by counting those

present in a measured amount and multiplying by the total volume.








- 10 -


Viability of the microfilariae was estimated by observing the rate of

movement and the percentage moving per low power (lOx) microscope

field. The microfilariac were then suspended in 50 ml. Krebs solution

and injected intravenously into the recipient dog over a period of ap-

proximately 10 minutes time.

Necropsy procedures and tissue preparation. The techniques des-

cribed by Jones and Gleiser (1954) for canine necropsies were follow-

ed. Tissues for histopathological examination were preserved in buf-

fered 10 formalin (pH 7.0). These were embedded, sections cut at

lO0 and mounted and stained with hematoxylin and eosin or vasson's

trichrome stain, as described by Thompson (1966).


RESULTS AND DISCUSSION

Natural infection with adult D. immitis. Fr'om necropsy examina-

tion of Id naturally-infected dogs, varying degrees of pathologic

lesions were found.

The primary lesions occurring in the heart and pulmonary arter-

ies included varying degrees of cellular changes ranging up to vil-

lous projections of the intima into the lumen of the pulmonary vessels.

In some cased of this series with villous projections of the intima

of the pulmonary arteries, endothelial proliferation in the smaller

arterioles was also seen. These microscopic lesions were also seen

accompanying the gross lesions of a roughened rugosese") appearance

of the lining of the pulmonary artery. Furthermore, this combination








- 11 -


of gross and microscopic leoioiu vas invariably fi'oue in dogs harboring

large numbers (30 or more) of adult D. immitis which usually extended

from the heart into the pulmonary arteries.

The hepatic lesions seen in this series of necropsies varied from

gross lesions of swollen, discolored livers to microscopic lesions of

advanced cavernomatous changes in the hepatic trinity. However, as a

general rule, the gross appearance of the liver was essentially normal,

but microscopic lesions were readily detected. These varied from cen-

trolobular congestion to cavernomatous formation in the hepatic trinity.

Specific lesions in other tissues from these naturally-infected

dogs were not detected. 1.Iicrofilariae vere invariably found in all or-

gans examined microscopically, but specific cellular changes attribu-

table to these larval forms could not be seen.

The lesions described from the present series are in full agreement

with the published accounts by Henningar and Ferguson (1957), Winter

(1959), Adcock (1961), and von Lichtenberg et al. (1962).

Perhaps the most plausible explanation of the lesions seen in con-

junction vith adult D. immitis infections is that of chronic irritation

by a foreign body. It is not difficult to accept this theory where one

sees large numbers of parasites in situ. Since much of the chronic fi-

brosis and sclerosis is irreversible, it also follows that the lesions

persist in the affected dog although the parasites may be removed (spon-

taneously, surgically, or by drug treatment). Furthermore, no doubt

these types of lesions may be seen in many other disease processes and








- 12 -


there is little evidence to indicate that any of them arc specific

for D. immitis infection. One exception to this statement may be

in lives changes, especially the "terminal li.ver failure syndrome"

described by von Lichtenbe-r; ut al. (3.962) and confirLmed by Savyer

and Veinstein (1963) which vill be discussed bulou..

T-ansfusion of non-infected dogs with D. immitis microfilariae.

The injection of approximately 3.5 million D. imilrtis microfi.ariae

into a non-infected dog resulted in the rapid onset of an anaphylac-

tic-like reaction (immediate vomition and defucation, muscle tremors

followed by clon'c spasms, rapid and shallow breathing, undetectable

carotid pulse, tetanic spasms of the muscles) and death. Attempts at

artificial respiration and the use of chemical. ;timulants were unsuc-

cessful and death occurred within 10 minuteL after the completion of

the injection of the .nic-ofilariae.

At necropsy, the g'oss lesions were those gcnexaclly ascribed

to anaphylaxis, i.e. edematous lungs, severe coigest'on of the 1'ver

and kidneys, etc. No adult or immature helm'.nth parasites were found

in the intestinal tract. Microscopically, the primary lesson was

that of centrolobular de eneaat'on and necroosij of the liver; a few

mici-ofilariae were seen in lung capillaries.

Thiu preliminary findinii of a "transfusion j.eaction" to a sus-

pension of D. immitis microfil.aiae is significant in that it may

indicate a role of circulating microfilariae in the canine heartworm

disease syndrome.










There can be several explanations given for this reaction, each

of which must be confirmed by additional experimentation.

First, the "transfusion reaction" may be a simple reaction to

foreign protein. Such a non-specific reaction could be initiated by

dead microfilariae present in the total injected number. No doubt

a percentage of the microfilariae uere adversely affected by the sep-

aration procedures in preparation for injection.

Secondly, the reaction could be due to prior sensitization of

the recipient dog with helminth antigens. This could be a signifi-

cant finding, if shown to be a consistent occurrence since it could

help to explain the apparent limitation of development of adult D. t,:

imiLtis in some dogs by an "interference phenomenon" between intes-

tinal or other helminths concurrently infecting the dog and infective

D. immitis larvae that were injected into it by a mosquito vector.

The same sort of phenomenon could explain some of the spontaneous

deaths of adult D. immrtis in dogs heavily infected with other hel-

minths. Of course, a main point for discussion would be to explain

how juch a cross-reaction could occur in only one direction, with

the effect exerted only on the D. immitis adults or larvae, if such

is the case.

A third explanation for the "transfusion reaction" may lie in

the basic nature of the dog's physiological and anatomical make-up.

According to Walker et al. (1960), the dog has one or more "hepatic

sphincters" that may be located in specific areas of the hepatic







- 14 -


venous system or may be, in fact, an innate ability of vasoconstriction

for its entire hepatic venous system.

Essex et al. (1931) observed an anaphylactic-like effect following

injection of helminth extracts into dogs, noting the similarity of the

reaction to that following injections of rattlesnake venom. Essex and

Thomas (1950) investigated the location of the "hepatic sphincter" in

dogs by using a vinyl acetate injection mass into the hepatic venous

system. They concluded that there were not sphincterss" as such, but

a general venous constriction in the dog liver following injections of

helminth extracts. They also pointed out that this phenomenon was much

more pronounced in the dog than in the cat, rabbit, or rat.

Roche e Silva and Grana (1946) reported that injections of Ascaris

sp. extract into the dog elicited an anaphylactic-like reaction. The

gross appearance of tho liver in such cases was that of extreme dis-

tention and congestion, indicating that the anaphylactic-like mecha-

nism was operating.

Grana (1947) reported confirmation of this phenomenon with either

Ascaris sp. extract of hydatid fluid both in vivo and in vitro, using

intact dogs or isolated, perfused dog livers, respectively.

These findings agree directly with the report of Ota et al. (1962)

who observed anaphylactic-like reactions in dogs receiving D. immitis

microfilaria-containing whole blood transfusions. Furthermore, our

findings in the present study lend further support to the proposal







- 15 -


that microfilariae are, in fact, etiological agents of some of

the pathologic changes in canine heartworm disease.

This line of reasoning on the development of liver patho-

logy is also in direct agreement with the proposed "terminal

liver failure syndrome" of von Lichtenbcrg et al. (1962). In

this report, the authors have postulated that the pathogenesis

of severe liver pathology described from their experimental

dogs vas primarily due to impaired circulation and chronic pas-

sive liver congestion. No doubt that impaired circulation may

contribute to the overall pathogenesis, but there is also evi-

dence from our preliminary study and from the other reports of

the effects of helminth extracts and microfilariae on hepatic

venous circulation that direct effects on the liver may also be

contributing to the clinical syndrome of heartworm disease. Con-

tinued studies are currently under vay in our laboratory to fur-

ther elucidate the mechanism of pathogenesis. It is hoped that

the improved method for obtaining microfilariae from infected

blood reported by Greenough and Buckner (1969) will expedite

such studies by providing larger numbers of microfilariae for

transfusion into experimental dogs produced in a carefully-con-

trolled (helminth-free) environment.











Acknowledgements

The author wishes to thank Dr. Ronald F. Jackson for help in

planning and execution of the research project from which this pa-

per has come. Thanks arc also due Dr. Robert H. Busch, .i.A.C.V.P.,

for help in necropsies and interpretation of histopathological ma-

terials. Also contributing to the success of the project have been

graduate students, Dr. Henry Carwile, Dr. T.K.S., kiukkur, and 1.ki.

David Weiner. The technical assistance of iir. Louis 2rgle, Mr.

lick Jattuso, fiirs. Lois Rogow, and ;-I. Jan Zvilling is also grate-

fully acknowledged.


- 16 -







- 17 -


Summary and Conclusions


The pathologic changes of canine heartworm disease described

by previous authors as attributable to adult D. immitis infection

were confirmed. These changes are thought to be due to the mechani-

cal irritation of the lining of the heart and adjacent blood vessels

which leads to the progressive development of fibrotic and oclero-

tic elements in the vessel walls. The latter changes may ulimate-

ly lead to other disease processes including elevated blocd pres-

sure, cardiac enlargement, pulmonary edema, and others.

The pathologic changes due to microfilaremia have been char-

acterized as similar to anaphylaxis. Although only preliminary

experimental evidence was available, correlation with published

reports on the hepatic venous circulation of the dog support this

theory. The microfilaremia that accompanies a population of adult

D. immitis in the heart and adjacent vessels in a dog may thus

play a role in the pathogenesis of canine heartuorm disease.










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TABL. I


Incidence of Dirofilaria immitis in Dogs from
Hillsborough County, Florida: 8 Samples (Feb.-Apr., 1968)


SUI.:ARY



1. Total examined 255

2. Total infected 74 (32.4,;)

3. Range of D. immitis county
in infected dogs:

1 10 : 49
10 20 : I6
20 50 : 4
50 or morc : 3

4. iean 12.1/dog




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