the effects of drugs oh experimental hepatic...

THE EFFECTS OF DRUGS OH EXPERIMENTAL HEPATIC CAPILLARIASIS IN MICE

Rosalind ^ranees Cheetham '#1

\S

A dissertation submitted to the Faculty of Science, University of

the Witwn tiers rand, Johannesburg in fulfillment of the requirements

for the Degree of Master of Science.

Johannesburg 1984

ABSTRACT

THE EFFECTS OF DRUGS ON EXPERIMENTAL HEPATIC CAPILLARIA3IS IN MICE

CIIEETBAM, Rosalind Frances* M.Sc.; University of the

Witwatersrand, 1984.

In this study fourteen anthelmintic compounds were tested in white

mice for their action against Capillaria hepatica (Nematoda).

Micej inoculated with 150 embryonated C. hepatica ova, were dosed

with the drugs on days fourteen to eighteen post inoculation. The

mice were sacrificed on day twenty-eight of the experiment* The

livers, where the C. hepatica ova are deposited, were deep-frozen

at -7Q°C to kill the eggs, and the number of eggs per gram of

liver was subsequently determined by means of the McMaster chamber

counting technique.

Of the anthelmintics tested, four prevented the deposition of

G» hepatica ova in mouse liver at low dose-level^. These were:

albendazole, 30.0 mg/kg; febantel, 30.0 mg/kg; mebendazole,

3.13 mg/kgj and oxfendazole, 12.5 mg/kg. Of these, mebendazole is

the only ons at present marketed locally for human use, and the

total dose administered is within the recommended dose range.

Scanning electron microscopy was used to study the effects of

these drugs on C. hepatica worms.

DECLARATION

I declare that this dissertation is my own, unaided work. It is

being submitted for the degree of Master o£ Science in the

University of the Witwatersrand, Johannesburg. It has not been

submitted before for any degree or examination in any other

University.

Rosalind "Frances Cheetham

day of 1984.

ACKNOWLEDGEMENTS

I gratefully acknowledge financial assistance Cor this study from

the South African Council for Scientific and Industrial Research,

the South African Medical Research Council and the University of

the Witwatersrand, I wish to thank the following pharmaceutical

companies for their donations of anthelu.intic drugs: Bayer* Ciba

Geigy, Coopers, XCI, MSI), Panvet, Salsbury and Smith-Kline.

I have received help a»d encouragement from many people during the

course of this work, and 1 am particularly indebted to Ms W. Meyer

for her photographic assistance and to Ms D. Els who typed the

first and second drafts of the manuscript. The final draft of

this dissertation was produced by Express Word Processing

Services, Benoni,

I am extremely grateful to Dr* A.N. Markus and Mr. W. Steinke who

translated Spanish and German publications, respectively, for me.

Finally I wish to thank my supervisor, Prof. M.B. Markus, for his

valuable assistance during the course of this work and his. / *

meticulous help in the preparation of this dissertation.

-iv-

CONTENTS

Page

ABSTRACT ~ i -

DECLARATION - ii -

ACKNOWLEDGEMENTS - iii -

TABLE OF CONTENTS - iv -

LIST OF TABLES - Vi -

LIST OF FIGURES - vii -

Chapter

1. INTRODUCTION 1

1.1 The Life Cycle of C, he pa I' lea, including

Comment on Spurious Infections 2

1.2 Reasons for study 9

2. HISTORICAL REVIEW 10

2*1 Drug Treatment for Experimental

C, hepatica Infection 10

2.2 Review of True Human Infections

with C. hepatica 13

3, MATERIALS AND METHODS 24

3.1 Experimental Animals 24

3.2 Supply of C . hepatica Eggs 24

3.3 Separation of Eggs for Bmbryonation 24

3*4 Etnbryoiiation 26

3.5 Egg Counting - for Embryonated Eggs 26

3.6 Experimental Design 27

3.6.1 Infection of Test Animals 29

3.6.2 Drug Administration 30

3 1,6.3 Modified Liver Digestion

Technique 33

i

iW iM «fc» inA i inni£ l i n n 'i Him

- V -

Page

3.6.4 Egg Counting - for Liver Examination 33

3.6.5 Evaluation of Results and Statistics 35

3,7 Scanning Eleeti*on Microscopy (SEM) 36

4. RESULTS 37

4.1 Effects of Drugs 37

4.1.1 Albendazole (Valbazen ® ; Figures 10-19 42

4.1.2 Febantel (Rintal © ); Figures 20-29 43

4.1.3 Mebendazole (Vertnox (§) ); Figures 30-33 43

4.1.4 Oxfendazole (Sysfcamex (E) ); Figures 34-43 44

4.1.5 Other Drugs Tested; Figures 44-53 44

4.2 Scanning Electron Microscopy (SEM) 68

5. DISCUSSION 72

5.1 Analysis of Material 72

5.2 Chemotherapy 75

5.3 Scanning Electron Microscopy (SEM) 79

APPENDIX A Details of Anthelmintics used 80

APPEKDIX B Results of Pilot Experiments 82

APPENDIX C Results of Low-dose Experiments 94

LIST OF REFERENCES 101

LIST.OP TABLES

Page

- vi -

TABLE 1 Summary of Results of Laitimlcr and Gruner

(1976) for Drug Treatment of C« hepatica

Infection 11

TABLE 2 True Human Cases of C. hepatica Infection 23

TABLE 3 Results of Pilot Experiments 38

TABLE 4 Results for Pilot Experiment Control Groups 39

TABLE 5 Results of Low-Dose Experiments 40

TABLE 6 Results for Low-Doae Experiment Control

Groups 41

TABLE 7 Lowest Effective Dose Against C» hepatica 76

* fra? 'Tgpgr*

LIST OF FIGURES

V J . l “

m ;

>r..

Page

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

1

2a

2b

2c

2d

3

4

5

6a

6b

7

8

9

FIGURE 10

FIGURE 11

FIGURE 12

FIGURE 13

14

15

lo

Life Cycle of C. hepatica

Light Micrograph of Sectional

C.« hepatica Eggs in Mouse Liver (x 400)

Scanning Electron Micrograph of

G, hepatica Eggs (x 1 000)


C. hepatica Eggs (x 2 000)


C» he pat: lea Egg - Polar Plug Region

(x 10 000)

Mouse Ear Marking Code

McMaster Slide

Dosing Syringe

Dosing Procedure - I

Dosing Procedure - II

Apparatus for Liver Digestion

Livers from Uninoculated Control Mice

Livers from Control Mice Infected with

C« hepatica

Livers from Mice Dosed with 1.88 rag/kg

Albendazole (ValMzen © )

Livers from Mice Dosed with 3.75 mg/kg

Albendazole (Valbazen © )

Livers from mice Dosed with 7,50 mg/kg




Livers from Mice Dosed with 30,0 mg/kg

Albendazole (Valbaaen © )

Livers from Mice Dosed with 60.0 rag/kg


Livers from Mice Dosed with 120 mg/lcg


6

25

28

31

32

32

34

45

45

46

46

47

47

48

48

49

: '

■

V 1 1 .1

Page

FIGURE 17

FIGURE 18

FIGURE 19

FIGURE 20

FIGURE 21

FIGURE 22

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

23

24

25

26

27

FIGURE 28

FIGURE 29

FIGURE 30

FIGURE 31

FIGURE

FIGURE

32

33

Livers from Mice Dosed with 125 mg/kg

Albendazole (Valbazert © )

Livers from Mice Dosed with 250 rag/kg

Albendazole (V-ubazen © )


Albendazole (Valbazen ® )

Livers Erom Mice Dosed with 1.25 mg/kg

Febantel (Rinfcal (g) )


Febantel (Rintal. CR) )


Febantel (Rintal © )


Febantel (Rintal ($) )


Febantel (Rintal )


Febantel (Rintal )


Febantel (Rintal (§))


Febantel (Rintal ® )


Febantel (Rintal (g) )


Febantel (Rintal (§) )


Mebendazole (Vermox 0 )


Mebendazole (Vermox © )





49

50

50

51

51

52

52

53

53

54

54

55

55

56

56

57

57

FIGURE

FIGURE

34

35

FIGURE 36

FIGURE 37

FIGURE 38

FIGURE 39

FIGURE 40

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

41

42

43

44

45

46

47

48

49

50

FlCURE 51


Oxfendazole (Systamex©)

Livers from Mice Dosed with 2,50 rag/kg

Oxfendaziole (Systamex © )


Oxfendazole (Systamex © )


Qxfendazole ( Systamex©)



livers from Mice Dosed with 25.0 mg/kg



Oxfendazole (Systamex®)

Livesrs from Mice Dosed with 56.25 mg/kg

Oxfendazole ( Systamex©)




Offendaxole (Systamex © )

Livei‘8 from Mice Dosed with 500 mg/kg

Amoscanate


Niclosamide (Lintex © )


Oxamniquine (Vansil © )


Oxyclozanide (ICI Liver Fluke Remedy © )


Pip'razine Adepate (Ascaradina © )

Livers from Mice Dosed with 500 "mg/kg

Piperflzine Citrate (Rid © )


Piperazine Dihydrochloride (Wazine © )


Praziquantel (Droncit ® )

- X -

Page

FIGURE 52 Livers from Mice Dosed with 500 mg/kg

Pyrantel (Combanfcrin © ) 67

FIGURE 53 Livers from Mice Dosed with 125 mg/kg

Rafoxanide (Ranide © ) 67

FIGURE 54 Scanning Electron Micrograph o£ a C, hepatica

Worm from an Untreated Mouse host (x 1 000) 69

FIGURE 55 Scanning Electron Micrograph of a C> hepatica

Worm from a Mouse Dosed with 3.13 mg/kg

Mebenda&ule (Vermox © ) (x 1 000) 70

FIGURE 56 Scanning Electron Micrograph of a C. hepatica

Worm from a Mouse Dosed with 30,0 mg/kg

Albendazole (Valbnzen © ) (x 1 000) 70

ITGUR1 57 Scanning Electron Micrograph of a C, hepatica


Oxfendazole (Systamex © ) (x 1 000) 71

FIGURE 58 Scanning Electron Micrograph of a C. hepatica


Febantel (Rincal © ) (x 1 000) 71

FIGURE 59 Graph to show the Linear Relationship

between Mouse Liver Weight and the Number

of Eggs per gram of Liver 74

IKTRODUCTfOK*«.»■«» • i irr«.t*- • •

Capillaria hepatica (Bancroft) 1893) Travossos, 1915 is

synonymic with both frichocephalus hepaticus and Hepafcicola' t m r t HMHmww ifw u y ,«ti> n »».»g« .-r*B'« ai«n»HM «m t i rrm mmu m tm **:*.......w ih h i t.ipw* •*•■■■■«>»

hepatica* It is in the same family as the well known

whipworm Trichuris triehiura. The classification of

C. hepatica is as follows (Chitwood and Chitwood, 1974)

Phylum

Class

Order

Suborder

Superfamily

Family

Subfamily

Genus

Species .

C< hepatica is parasitic in the liver of a wide range of

animals, including rats, mice, dogs, muskrats, beavers,

rabbits, peccaries, tnonkeys, squirrels, pigs and cats

(Baylis, 1931; Luttemoser, 1938b; Faust, 1949). It is

cosmopolitan in its distribution and there are several

reports of C. hepatica from mammals in the Republic of South

Africa and elsewhere in Africa, The infection rate varies

but it is undoubtedly highest in rodents,, For example, Shorb

(1931) records an infection rate of 48 per cent amongst brown

rats Ra t* tus norvpf*i eus in Baltimore, United States of

America. In the same area, Luttermoser (1936) found

C, hepatica in 86 per cent of adult R. norvegicus and 22 per

cent of juveniles, Cochrane _et_ a_U (1957) recorded the

parasite in 28 per cent of a random group of rats caught in

the vicinity of Johannesburg, Republic of South Africa,

Waddell (1969) found 79 per cent of a group of R. norvoglcus

from Brisbane, Australia to be infected with C. hepatica.

More recently a survey in Connecticut, United States of

Neroatoda

Aphasmidia

Snoplida

Dorylaimina

’frichuroirica

Trichuridae

Caplllarinae

Capillar!a

Capillaria hepatica

America by Conlogue ct al. (1979), revealed that 81 per cent

of the R. norvogicus rats examined were parasitised by C.

hepatica, Amongst other host species the infection rate

would appear to be lower. For example, Wright (1930) records

G, hepatica infestation in only one per cent of the dogs he

examined and McQuown (1954) reported it from 4 per cent of

squirrels trapped in Louisiana, United States of America.

The Li fo Cycle of C. hepatica, including Comment on Spurious

Infections

The life cycle of C. hepatica is summarised in Figure 1. The

adult worms are found in the liver, where the females lay

their eggs. 0. hepatica worms are of typical trichurid

appearance, having a thicker posterior portion containing the

intestine and reproductive organs, and a whip-like anterior

portion containing the stichosome, oesophagus and bacillary

bands. According to Neafie _et_ al. '1976) there is

considerable variation in body size of the females, their

length varying from 52 ram to 104 mm and width from 78^pn to

184 jim. Males are reported to be more uniform in size,

namely in the region of 22 mm long (Neafie _et al,* 1976).

However, Luttermoser (1938b) states that he found males which

and ?8^im in

width. It must be stated, however, that it is extremely

difficult to measure the length of C. hepatica worms as the/

lie tightly coiled within the- livei- tissue and are very

difficult to dissect out.

After mating, the female worms produce many thousands of

eggs. These are not excreted from the host’s body but remain

within the liver until the animal dies and the eggs are

released into the soil as a result of decomposition.

Alternatively, if the host is eaten, the eggs pass unaltered

and unharmed through the digestive tract, to be excreted with

the faeces (Shorb, 1931). A good example of the latter

method of egg release is cannibalism amongst rats, which is

measured up to 37 tnm xn length and between 26^im

Larvae, mature,

mate and lay eggs

Adult worms and eggs

in liver of mammalian

host, e.g. rat

VHost eaten by

carnivore

VEggs pass unaltered

through the intestine

and out in the faeces

Larvae hatch in

intestine and move

to the liver via the

hepatic portal vein

Embryonated eggs

ingested by new

hos t

Death of host

Decay of body and

release of eggs

The eggs embryonate

under correct

conditions of

oxygen, temperature

and moisture

FIGURE I Life Cycle of C. hepatica

thought to account for the high prevalence of this parasite

within rat populations (Calle, 1961; Farhang-Azad, 1977). It

is also possible for humans to ingest unombryonated ova of G .

hepatica, for example, by eating the liver of infected wild

game (Brosius et al, 1948; McQuown, 1950). The occurrence of

C. hepatica eggs within the host as a result is teamed a

spurious infection and, although it may cause transient

symptoms of pain and diarrhoea, it is not a long-term

infection of the liver. It is probable that the prevalence

of such spurious infections is greater than presently

thought, because of the similarity between the eggs of C»

hepatica and T> trichiura (McQuown, 1950). Both these

species have the same basic nematode eggshell structure, that

is, there is an outer vitelline layer, a middle chitinous

layer and an inner lipid layer. In C» hepatica, the inner

portion of the chitinous layer is helicoidal whilst the outer

portion forms a "pillar and beam-like" network which is not

present in T. trichiura (Grigonis and Solurnon, 1976; Warton,

1980). This unusual structure in the chitinous layer of the

ova of G. hepatica serves to give the outer layer, of what

appears to be a double shell, a striated appearance not seen

in T. trichiura. The shape of the eggs of C. hepatica

an<* T y trichiura are similar but closer inspection reveals

that while both ova are elliptical, those of C. hepatica are

larger, measuring 54 j m to 65 jim by 29 ^im to 33 jam ' »

that 49 jam to 54 jim by 21 jam to 23 j m as in T. trichiura.

The ova of both species have polar plugs but

whereas in C. hepatica they do not protrude beyond the outer

shell layer (see Figures 2a, 2b, 2c and 2d), they do in

T. trichiura. It should also be noted that the C. hepatica

ova are passed in the faeces wlu-n in the morula or immature

two-cell stage. T. trichiura ova, on the other hand, are

always passed while still unsegmented (Calle* 1961).

.O T P

thought to account for the high prevalence of this parasite

within rat populations (Calle, 1961; Farhang-Azad, 1977). It

is also possible for humans to ingest unerabryonated ova of Cj_

hepatica, for example, by eating the liver of infected wild

game (Brosius et al, 1948; McQuown, 1950). The occuirancs of

C. hepatica eggs within the host as a result is termed a

spurious infection and, although it may cause transient

symptoms o£ pain and diarrhoea, it is not a long-term

infection of the liver. It is probable that the prevalence

of such spurious infections is greater than presently

thought, because of the similarity between the eggs of C.

hepatica and T. trichiura (McQuown, 1950). Both these

species have the same basic nematode eggshell structure, that

is, there is an outer vitaltine layer, a middle chitinous

layer and an inner lipid layer. In C. hepatica, the inner

portion of the chitinous layer is he^ *dal whilst the outer

portion forms a "pillar and beam-li. tetwork which is not

present in T» trichiura (Grigonis and Solomon, 1976; Warton,

1980). This unusual structure in the chitinous layer of the

ova of C, hepatica serves to give the outer layer, of what

appears to be a double shell, a striated appearance not seen

in T* trichiura. The shape of the eggs of C. hepatica

and T. trichitira are similar but closer inspection reveals

that while both ova are elliptical, those of G. hepatica are

larger, measuring 54 jum to 65^pm by 29 jam to 33^jim rather

that 49 Jim to 54 jim by 21 pm to 23 jira as in T. trichiura.

The ova of both species have polar plugs but

whereas in C. h epatica they do not protrude beyond the outer

shell layer (see Figures 2a, 2b, 2c and 2d), they do in

T. trichiux- , It should also be noted that the C. hepatica

ova are passed in the faeces when in the n,orula or immature

two-cell stage* T. trichiura ova, on the other hand, are

always passed while still unsegmented (Calle, 1961).

FIGURE 2a Light Micrograph of Sectional G. hepatica Eggs in

House Liver (x 400)

(P - Polar Plug, C - Chitinous layer)

(x 1 000)

(P - Position of Polar Plug)

\

PIGURE 2c Scanning Electron Micrpjn^p h ^ ^ ^

(x 2 000)

(p - Position of Polar Plug)

FIGURE 2dScanning: F.lectrow Hicroaraph o£ C y Hp.p^j££jEgRS.

Polar Plug RepiimL. (x 10 000)

(P - Position of: Polar Plug,)

& v.

Taking account o£ the above features, differentiation between

the ova of these two helminth species should be

straightforward.

Once free in the soil, the eggs must embryonate before they

are infective. The factors which determine the rate of

embryouation are oxygen and moisture availability and the

ambient temperature, As early as 1931, Shotb experimented

with embryonating C . hepatica ova at temperatures of 22°Cj

30°G and 37.5°C for periods of twenty-five and forty-two

days. His results indicated that although many of the ova at

37.5°G had embryonaled by day twenty-five, most had

degenerated by day forty-two. Those at 22°C and 30°G,

although slower to embryonate, did not degenerate. Wright

(1961) confirmed these findings, determining the embryonation

time at 20°C to 24°C to I : a maximum of eight weeks. He also

found that embryonation did not occur in eggs which had been

previously subjected to a temperature of -40°C for a period

of twenty-two hours.

Embryonated ova, when ingested by a new host, hatch in the

intestine and penetrate the intestinal mucosa from six hours

after hatching (Luttermoaer, 1938b). The larvae enter the

hepatic portal vein and are carried to the liver. Wright

(1961) studied the development of C. hepatica larvae and

found that second stage larvae were present in the liver

three to seven days after infection, that third stage larvae

were apparent from day five and that fourth stage larvae were

present from day nine. The male larvae matured faster than

the females, being considered fully developed at day

eighteen, but females were not mature until two days later*

Wright (1961) records first seeing female worms bearing eggs

on day twenty-one, while Luttermoset (I938h) reports seeing

female worms containing eggs on day eighteen in mice and on

day twenty-one in rats,

It would appear that animals can withstand very large doses

of C. hepatica ova, the number of eggs ingested being of more

critical importance in juveniles. Luttermoser (1938b) found

that adult mice could withstand doses of 1 000 eggs, although

the development of the worms was slower than normal and they

were smaller. Immature mice, however, were killed by doses

of 500 eggs, Similarly, Luttermoser (1938b) demonstrated

that a lethal dose in juvenile rats was between 2 500 and

5 QQ0 ova, whereas adults could tolerate doses of 30 000 ova.

In animals with massive infections, worms were found free in

the body cavity, where they appeared to mature normally. In

addition, worms were infrequently found in the lungs, but

here the worms lived only eighteen days. Luttermoser (1938b)

demonstrated that a heavy infestation with C . hepatica caused

retarded growth of the host, and, especially in mice,

emaciation. It would appear from his results that in

rats a small initial infection of C. hepatica "immunised"

the animal against a larger challenge infection given later.

Zahner et al. (1980) pursued this idea in some work using the

multiraammate mouse Mastomys natalensis as the host animal.

They determined that an initial infection of C. hepatica did

suppress the reproductivity of a large, but sublethal,

challenge infection, but if the challenge infection was only

of moderate size then the existing infection had no effect on

it.

Normal egg production increases rapidly from about day twenty

of the infection to a peak at day forty, with no further egg

production after about day seventy (Lammler et al., 1974).

After their reproductive functions have been completed, the

C. hepatica worms die and slowly disintegrate.

9

1.2 Reasons for Study

The present investigation has been prompted by the fact that

there are thirteen cases of Q. hopatica infestation in humans

documented in the literature to date (see Historical Review).

Three of the cases occured in South Africa. Over half of the

thirteen cases resulted in death, the infection either having

been discovered at autopsy*, or, even though the infection was

diagnosed by liver biopsy, the patient died due to lack of

suitable treatment.

There is no established cure for C. hepatica infection in

roan. Some German researchers have looked at the effects of a

number of drugs on C. hepatica in M, natalensis (Lammler and

Gruner, 1976), but it was thought that there was scope for

wore work, in view of the production of new anthelmintics.

" t . ‘v ^

10

2. HISTORICAL RKVTF.W

2.1 Drug Treatment for -Experimental C, hepatica Infection

The first report of experimental chemotherapy of C. hepatica

infection is given by Wadde.il (1969). He treated rats Rat tug

rattus subcutaneously and intraperitoneally with three

200 mg/kg doses of methyridine on days fourteen to sixteen of

the infection. The results of this study indicated that

eviposition had been almost totally prevented,

The second study was carried out using the multimammate rat

Hastomys natalensis (Lammler and Griiner, 1976). Their

results are summarised in Table 1. These authors examined

twenty"four compounds for activity against C, hepatica by

giving five doses, one on each of days fourteen to eighteen

of the infection, Fenbenda-jole, mebendazole and parbendazole

were the most effective drugs at low dose levels, as

indicated by the high percentage reduction in the egg counts

as compared to the control groups. These three drugs were

tested further against stage ttfb and stage three larvae - by

dosing on days five to nine of the, infection. Mebendazole

and parbendassolc were proved to be most effective.

■*Jk __A __-— v f - W .UL. f t -

TABLE I Summary of Results of Ltfnunlor and Griiner (1976) for Drug

Treatment of C. hepatica Infect i on.

DRUG DOSE mg/lcg x 5*

— —------% REDUCTION

IN EGG COUNTS 1

Bitoecanate p . o . ^ 20 040 0

Cambenda^ole p . o . 1.56 03.12 56.26.25 99.012.5 98.625 10050 100100 100

Diethylcarbaiuazine p . o , 200 0400 32.3

Disophenol s .c. $ 10 020 0

Dithiazanine p ,o . 40 9.4

Fenbcndazole. p.o. 1.56 0

3.12 33.8

6.25 93.612.5 99.525 10050 99.9

100 100

Fentln'one p.o. 20 17.0

HOE 28637a p.o. 200 1.7400 0

HOE 33258d s.c. 2.5 05 31.1

10 83.320 99.820 10040 100

Hycanthone s.c. 10 19.1

Levamisole p.o. 10 015 43.620 98.840 99,3

TABLE 1 (Continued)

DRUG DOSE % REDUCTIONmg/kg x 51 IN EGG COUNTS1

Mebendazole p.o, 0.78 01.56 35.23*12 89.56.25 99.98

12.5 10025 100

100 100

Methyridine s*c. 50 0100 0200 84.2400 100

... ~ -..lT1........... .......... .............. ............... .............

Metrifonate p.o, 200

m n n r . r m . M M W inn . rr , , , ^

15*1400 33.5

Morantel Tartrate p.o* 200 0400 0

Niridazole p,o» 100 0

Nitrofurantoins p.o* 100 14.9

Nitroxynil s.c. 5 5*3'0 58,9;o 62*: ik 0 12,6 ]

Parbenda?.ole p.o. 1 . %3*32 13.96.25 56*112.3 83*925 98.250 99*6

100 100200 r o

Fiperazine Citrate p.o. 250 0500 0

Pyrantel Tartrate p.o. 100 0200 0

Suramine s.c. 20 040 43.3

Tetramisole p,o« 20 030 95.340 99*7

TABLE 1 (Continued)

DRUG ' DOSE mg/kg x 5-*-

% REDUCTION IN EGG COUNTS1

Thiabendazole p.o. 12.5 025 61.750 34.4

100 77.5200 87,2300 98.1400 99.95500 99.97

1 See text2 p»o, per rrnlis3 s.c. subcutaneous

Review of True Human Infections with C. hepatica

The first report of a ttue case of C . hepatica infection in

man was recorded by Dive et al, (1924)* The case was that of

a twenty-year-old man who had been serving in the array in

India. He exhibited the symptoms of septic pneumonia, and

after his death the autopsy revealed abcesses in the lungs

and the liver. Microscopic examination of tissue in

thevicinity o F the liver abcess revealed masses of eggs which

were identified as Kepaticola hepatica (a synonym for

C. hepatica). Material from the periphery of the liver

lesion revealed mature female, worms. Ho such eggs or worms

were recovered from the l*ing tissue. It was concluded that

pyaemia, resulting from the nematode infection and the

secondary lung infection, was the cause of death.

The next description of a true human infection of C. hepatica

was given by McQuown (1950) after a girl aged seventeen

months had been admitted to hospital in Louisiana in the

United States of America, thought to be suffering from

pneumonitis and possibly meningitis. When she was first seen

as an outpatient t/he was suffering from a nasal infection,

cough and dyspnoea with a raised temperature. She was

treated with sulfadiazine but three days later she was

admitted to hospital acutely ill, apathetic, stuporous and

with a grunting respiration. Her liver was enlarged. The

patient was placed on routine critically ill care but died

twenty-six hours after admission. At potft-mortem the heart,

lungs, spleen, kidneys and liver were seen to be enlarged.

The appearance of the liver was abnormal, being yellowish-

brown. When cut, the liver proved to be soft with numerous

small haemarrhagie areas and greyish-tan areas 1 mm to 3 mm

in diameter scattered throughout the parenchyma and

resembling small focal areas of necrosis or abceases.

Microscopically, the liver was found to contain numerous

granulomas filled with C. hepatica ova and located in the

periportal spaces. The surrounding chronic inflammatory

reaction encroached on, and in some places, destroyed, the

liver lobules. No adult parasites were seen. The lungs did

not contain parasites. Ascaris lumbricoides nematodes were

found in the intestine. The final pathological

diagnosasware; C. hepatica infection of the liver, pulmonary

oedema, interstitial pneumonitis, acute cardiac dilation with

congestive failure, malnutrition, dehydration and ascariasis.

Otto et_ al. (1954) documented the case of a seven-year-old

girl whose C. hepatica infection was diagnosed b'» liver

biopsy, but who subsequently died. This girl suffered from

sickle-cell anaemia and had been admitted three times

previously for sickle-cell crises, before being a d m u e d to

hospital in Maryland in the United States of America in 1951

with fever, shortness of breath and nose bleeds of six days

duration. In addition to these symptoms, anorexia, lethargy

and severe malaise were, evident after admission and she

vomited clotted hlood. She. later developed headaches and

epigastric, scapular and neck pains. The liver and spleen

were found to be enlarged. On the forty-sixth hospital day a

laparotomy was performed. The spleen x*as seen to be twice

its normal size and the liver was enormous. The liver

surface was smooth with purplish and white speckles but

without gross scarring. A liver biopsy and lymph node were

taken. Grossly, the liver revealed tiny yellow foci 1 nun in

diameter scattered evenly throughout. Microscopically, it

could be seen that the normal liver architecture had been

completely disrupted by the parasitic infection. Adult

worms, often containing eggs within their body cavities, were

usually at the centre of the necrotic foci, which were

surrounded by an intense inflammatory response. It was

thought that these lesions were probably located in the

portal areas3 but because of the disruption of the liver

structure j the authors could riot be certain. Eggs were

present in the liver but no embryonated eggs were seen, which

is characteristic of G. hepatica infection. The treatment of

this patient was largely supportive and initially consisted

of administration of vitamin D and calcium, The liver

remained enlarged and two courses of chloroquine were given.

Chloroquine was chosen because its hepatotoxicity is low;

and it is known to be heavily deposited in the liver and is

active against some helminths. This treatment was completed

on the 107th hospital day but the liver continued to enlarge

and on day 168 a second liver biopsy was performed. This

second biopsy revealed tiny grey foci which were seen to be

broad areas of scarring containing enormous numbers of eggs.

Again the eggs were thought to be, concentrated in the portal

areas. The liver remained enlarged and the patient was

discharged on day 203, to be readmitted twice more (orv days

307 and 331 after the initial admission), in an unchanged

condition. Nearly two years after the onset of the illness

the patient was chronically ill and was readmitted for

weakness, nosebleeds and anorexia. No treatment was given

and two mont! 9 later she was admitted to hospital with acute

fever, She developed convulsions and suffered respiratory

arrest and died five minutes after admission. At autopsy the

heart, thymus, spleen and kidneys were enlarged, the liver

being grossly enlarged at nearly four times Khe normal

weight. Histological examination of the liver supported the

previous observations which identified the parasite as

C. hepatica. The authors state that one lung section showed

possible evidence of C. hepatica infection in the form of

hyaline and calcified bodies in giant cells, although no

actual eggs or worms were seen. The final diagnosis in this

patient was C, hepatica infection of the liver with

granulomatous lesions containing eggs and worms, cirrhosis of

the liver, ascites, granulomatous lesion of the lung with a

foreign body reaction, a history of sickle cell anaemia with

repeated crises, sickle cell lesion of the spleen with

splenemegaly, marked intravascular sickling of red cells,

foci of enc eph aIema1ac i a , brain, cardiac hypertrophy and

dilatation; possible rheumatic myocarditis with Aschoff body

formation; renal hypertrophy with glomerular engorgement,

slight focal calcification of renal tubular ephithelium. In

the final analysis it was admitted that the sickle cell

disease contributed to the child’s illness and hepatic

dysfunction, The hepatic disruption was, however, wholly

attributed to G< hepatica, but hepatic damage, known to occur

in sickle cell anaemia, may have further aggrevated the

symtoms of hepatic insufficiency,

Turhan et al. (1954) published the fourth case of true

C. hepatica infection. In this instance, C, hepatica was

found incidentally at autopsy in a sixty-year-old man. Thf

clinical findings were bronchopneumonia and senile dementia,

and the pathological findings were bronchopneumonia and

C, hepatica in the liver.

The fifth case of true human infestation with G. hepatica was

diagnosed in Honolulu and reported by Ewing and Tilden

(1956). This was the case of a fifteen-month-old female

infant who was first seen by a physician because of fever,

anorexia, cough, constipation and a reluctance, to walk. The

parents stated that their daughter had been known to eat

dirt. An ascarid infection was diagnosed and treated with

two courses of hexylresorcinal, which in turn required

Benadryl © (diphenhydramine HCL) to treat the side effects.

Terramycin ® (oxytetracycline) was prescribed for the fever

16

17

but upon failing to respond to this treatment, and also in

view of the pulmonary congestion, the child was hospitalised.

Fourteen days after the patient was first examined a course

of Atabrine © (quinacrine) was administered. At about the

same time the liver and spleen were found to be enlarged*

Shortly after this a course of Hetrassan ©

(diethylcarhamazine), an effective treatment against

ascariasis and filariasis, was given together w,, Lederplex

® , a brand of vitamin B complex. Nearly one month after the

girl was first examined a liver biopsy was performed. The

liver was seen to be greatly enlarged and the surface was

studded with pearly white granulomatous lesions up to 2 mm in

diameter. Microscopically, the liver showed massive

deposition of ova in the portal areas together with many

worms, and an associated inflammatory reaction. The parasite

was identified as G. hepatica. The patient w s discharged,

although the liver and spleen were still enlarged. Three

months after the initial examination, the patient was

readmitted to hospital with bronchopneumonia, cough and

dyspnoea. The liver and spleen were further enlarged.

Treatment consisted of continous oxygen, penicillin and

streptomycin, blood transfusion, intravenous and subcutaneous

fluids and parenteral vitamins. However, the child died a

week later. At necropsy the spleen was enlarged and the

liver greatly enlarged, microscopical examination of the

liver revealing the ova previously identified as

C , hepatica. However, the worms had by now degenerated.

There was considerable liver cell destruction towards the

periphery of the lobules and the resultant fibrosis had

distorted the normal liver architecture considerably.

Microscopic examination of both the lungs and kidneys

itvealed calcified material, which it was thought provided

presumptive evidence of 0. hepatica. parasitism in these

organs, but no eggs or worms were seen.,

Cochrane et al. (3957) provide an account of the sixth case

of G. hepatica in man to be reported in the world, and the

first case from South Africa. The patient in this instance

was a fifteen-month-old girl who was reported to be an earth-

eater. She was admitted to hospital in Vanderbijlpark,

Transvaal, seriously ill with fever, umbilical hernia,

enlarged glands and enlarged liver. A liver biopsy was

performed approximately two months after the onset of the

child's illness. The liver was found to be moderately

enlarged and the surface studded with pinhead-sized yellow-

grey nodules, in places aggregated to form an irregular

lesion of 2 cm to 3 cm in diameter. The spleen was not

enlarged. Microscopically, large numbers of ova were seen in

the portal tracts. The ova were surrounded by areas of

inflammation, which had resulted in distortion of the liver

architecture. The parasite was identified as C. hepatica*

Non-specific drugs which were given included Terramycin ©

(oxytetracycline), Achromycin ® (tetracycline with sodiutn-

metaphosphate), penicillin, Chloromycetin © (chloramphenicol),

Meticorten©(ptadnisone) and Methischol®(choline dihydrogen

citrate)* As specific therapy against the parasitic

infection an antimony! drug, Triostam ® (sodium antimonyI

gluconate), was given. This controlled the temperature and

the liver enlargement decreased, Approximately two~and-a-

half years after the first liver biopsy, a second one was

performed. The liver size and surface were deemed normal.

No ova were seen on microscopic examination, although some

periportal fibrosis was present. The haematological findings

were also normal. Neatly four years after this child was

first examined Cochrane and Skins tad (1960) published a

follow-up report.

The seventh published case of human infestation with

C, hepatica (Ward and DGtit, 1959) describes the illness of a

two-year-*old girl. Her case history had included diarrhoea;

and stool examination had revealed ova of A. lumbricoides and

T. trichiura, for which infections she. was treated, However,

the diarrhoea persisted and she developed cough, fevei and

nausea with vomiting. Prior to hospital admission she had a

19

convulsion,, On admission to hospital in New Orleans ir. the

United States of America, she was lethargic, dehydrated and

suffered from dyspnoea. The patient was treated with

intravenous fluids, antibiotics, antipyretics and sedatives.

About eight hours after admission she expired, having

received treatment for respiratory distress. The clinical

diagnoses were bronchopneumonia, intestinal parasitism and

gastroenteritis due to Shigella sonnei, with anaemia and

dehydration. Autopsy revealed that the colon was infected

with T. trichiura and that the lungs and liver were enlarged.

The liver was tan irt colour and, when cut, tiny scattered

foci of greyish-tan discolouration about 1 mm in diameter

were. seen. Kictoscopically these proved to be fibrous

granulomas enclosing numerous ova of C. hepatica in the

periportal areas. Microscopic examination of the lungs did

not reveal any parasites or gram; iomata. Additional

diagnoses of interstitial pneumonitis and 0. hepatica

infection of the liver were then made. The authors believe

that the C . hepatica infection was an incidental finding and

that death was due to bronchopneumonia and gastroenteritis,

although, it would seem reasonable to assume that the

debilitating effect of C. hepatica infection must have

contributed to the patient's death.

Calle (1961) describes the eighth genuine case of human

infection with C, hepatica. The patient was a twenty-month-

old male infant who was admitted to hospital in North

Carolina in the United States of America, with fever,

lethargy and abdominal swelling. He was known to 'm, a dirt

eater. He was treated for sore throat and infected ears but

there was no reduction in the fever. On the' seventeenth

hospital day a liver biopsy was done. The liver was seen to

be purple-tinted, indurated and mottled, with numerous grey

scars scattered throughout tlve organ. Microscopic

examination of the scars revealed that they were granulomata

situated in the portal areas of the liver and containing

C. hepatica ova. These granulomata were surrounded by an

inflammatory response which had caused partial disruption of

the liver architecture, Partially disintegrated worm

fragments were seen but there was no calcification. The

infection was treated with Delvex (§) (dithiazanine iodide).

The patient improved and the liver became smaller. Although

liver biopsy was not performed again, the child appeared to

be in good health ten months after the initial illness.

The ninth case report is also the second in South Africa.

Kallichurum and Blsdon-Dew (1961) describe the case of a

five-year-old girl who was admitted to hospital in Dnrban,

Natal, with measles, bronchopneumonia and dysentry. Stool

examination revealed T, trichiura, Trichomonas hominis and

A. lumbricoides. There was apparently no clinical evidence

of liver disease and no blood count was done* The patient

died on the sixth day. At autopsy the liver was seen to be

of normal size bxxt studded throughout with gtitty yellow-

white specks 1 tran to 2 mm in diameter. Microscopically these

appeared as foci of fibrosis and inflammation containing

numerous ova of C> hepatica, but the surrounding liver

architecture did not appear distorted.

Romero Garcia et al. (1962) provide the tenth report of human

infestation with C, hepatica. The case is chat of a twenty-

two~month old female child from Guadalajata in Mexico, The

family's living conditions were fairly primitive and there

were dogs, cats and chickens close by which often strayed

into the house. Rats and mice were also pt-esent, The child

was known to be an earth-eater. For two months prior to

hospital admission the child's symptoms had included fever,

sweating, anorexia, vomiting, oedema, pale skin,

palpitations, severe headaches, irritability, and loss of

weight. Immediately prior to admission the mother had

detected abdominal swelling and a mass in the abdomen. At a

previous hospital, blood tests had been carried out and

leukaemia diagnosed, Upon admission, for the second time to

hospital, the general clinical picture was as already

described, and, additionally, the liver was found to be

enlarged and painful to the touch. Blood tests and liver

function tests revealed leucocytosis, eosinophilia and

bypergammaglobulinaemia. Several intestinal parasites were

found. These findings did not confirm the dir^nosis of

leukaemia. Hetrazon ($) (diethylcarbanmine) was administered

to deal with the parasitic infections and a steroid was also

given in varying doses, from 2 mg to 15 mg per day. The

steroid resulted in the fever disappearing and the

hepatomegaly, eosinophilia and leucocytosis being reduced.

If the steroid therapy was suspended the fever returned and

the patient's general condition deteriorated. Twelve months

after initiation of treatment, the steroid administration was

stopped with no ill-effects. At this time a liver biopsy, by

means of a laparotomy, was performed. The liver was seen to

be enlarged, with many whitish-yellow nodules of 1 mm to 2 mm

diameter scattered over the whole surface of the organ.

Histology revealed numerous granulomata in the portal spaces.

The granulomata contained eggs of C. hepatica. Fibrotic

areas were present. There was no infiltration in the lungs.

The patient was thus diagnosed as having an infection with

G. hepatica. No further treatment for the infection was

given.

Details of a case of human G. hepatica infection in Italy

were published by Cislaghi and Radice (1970), A forty-month"

old girl who had been ill intermittently for about two ye<u*s

was admitted to hospital, chronically ill. The major finding

waa liver enlargement and a liver biopsy was performed. The

liver appeared cirrhotic with numerous grey scars scattered

throughout the organ. Microscopically these areas were seen

to be granulomafca cont,nixing ova of 0. hepatica, which were

partially calcified. There was an inflammatory response but

no adult worms were seen, The treatment is not detailed

beyond the use of unspecified anthelmintic drugs, but the

patient appears to have survived,

22!

Silverman at_ ajL (1973) give ar account of the infestation of

a seventeen-month-old infant girl from Bethal in the

Transvaal. This constitutes the twelth case in the world

literature and the third in South Africa, The child was

admitted to hospital after suffering from cough and fever for

a month. She was known to be a sand-eater. The liver and

spleen were enlarged and percutaneous liver biopsies were

taken. Histological examination showed numerous granulomata

surrounded by inflammation and containing ova which were

identified as C. hepatica. Apart from these features the

liver parenchyma appeared normal. Treatment proceeded wi»'h

oral iron for anaemia, and Hetrazan (§) (diethylcarbamazine)

against the parasitic infection. As the patient remained

ill, Triostarn © (sodium antimonyI gluconate) was

administered. This led to a general improvement in health.

The patient was discharged. Four months later, a repeat

biopsy revealed reduction of the fibrosis but ova were still

present. One year after the initial admission the child was

still well.

The final and most recent case to be reviewed is from Kaduna

in Nigeria and is described by Attah et_ al. (1983). A

tw^nty-seven-year-old woman complained of increased swelling

in the right side of the abdomen for the previous two years.

The liver was found to be markedly enlarged. Percutaneous

liver biopsy revealed extensive fibrosis and inflammation

surrounding ova Of C. hepatica in the portal areas. There

was some granulomatous reaction but it was not widespread.

There was no calcification and no adult worms were seen.

Treatment with potassium iodide na an outpatient was planned

but the patient was lost to follow-up, Presumably she. is

still alive,

The true human cases of C, hepatica infection are summarised

in Table 2.

t

,-Vt

23

\

do*HWuf ilweHrtu•H«wPM<u.c

*1

oIW003ft)w«o

B)

<N

3

> *s' a .

Experimental Animals>i I ..........H I rl'i' W

All the animals used for experimental purposes were male

Swiss-Webster White mice obtained from the Central Animal

Unit at the University of the Witwatersrand, The mice

usually weighed between 20 g and 33 g at the start of an

experiment, The mice were housed in groups of five in solid-

bottomed plastic cages with sawdust as bedding, and fed a

diet of mouse cubes and water ad libitum. Every experimental

mouse was given an individual number by means of ear

punching, using the code shown in Figure 3.

Supply of G. hepatica Eggs

Initially egga of 0. hepatica were obtained by trapping black

rats Rattus rattua in the vicinity of Johannesburg by means

of 1 •'rge metal rat traps, killing them with ether and

examining the liver of each animal for the characteristic

fibrosis caused by C. hepatica, * When an infected liver was

found it was processed as in sec ions 3,3 and 3.4 and mice,

as in section 3*1* were then infected with the parasite, as

in section 3,6,1, Once the infection had been established in

the laboratory it was maintained by routine passage in white

mice,

Separation of Eggs for Embryonation

The infected liver was cut into small pieces and then

pulverised using a pestle and mortar, in order to break open

any fibrotic masses containing eggs. The resulting mixture

was then flushed through a piece of fine gauze material with

tap~water in order to remove as many large liver particles as

possible. The filtrate was kept at 4°C and allowed to settle

Tens Units Thousands Hundreds

"N

V J ?"N.

60

U °

L\

01

• 0

25

r 7000

(^ 8 0 0 0

9000

FIGURE 3 Mouse. Es- Mnrkinp, Code— .............. . . m.'iiiw......... . ........ ..I . . . r . . U r m i m m .................. . - r - . . . . -

(By courtesy of the Department of Toxicology and Reproductive

Studies, VJyeth Laboratories Ltd., Maidenhead, Berks;., U.K.)

for at least twelve hours. The supernatant was sucked off

using a venturing pump, leaving the eggs, which sink in

water, in the sediment at the bottom of the container* The

container was then refilled with tap~water and replaced at

4°C. In order to wash the eggs as thoroughly as possible,

the process of sedimentation and removal of the supernatant

was repeated several times over a period of approximately

seven days, until the supernatant was clear.

3.4 Emln*yonation

The clear supernatant of the washed eggs was discarded and

the eggs, in a minimum of tissue debi'is, were placed in a

petri dish containing a small amount of tap-water, which had

been allowed to stand for at least two days. The petri dish

containing the eggs was then placed for six to eight weeks in

an incubator maintained at a temperature of between 27°C and

3G°C (Shorb, 1931; Luttermoser, 1938a; Wright, 1961;

Vollerthun, et al., 1974; Lammler and GrUner, 1976; Zahner et

al., 1976). During this time the egg^ were aerated two to

three times per week by means of a Pasteur pipette and

dftchlorinated water was added as required, so that the eggs

did not dry out. After six to eight weeks the percentage

embryonation was determined by microscopic examination of the

eggs.

3.5 Egg Counting - for Embryonated Eggs

A modified McMaater counting technique was used (1. >.i*.nr.y of

Agriculture, Fisheries and Food, 1971; Vollerthun et al.,

1974; Lammler and GrUner, 1976; Dunn, 1978). A known volume

of eggs in solution was added to zinc chloride solution with

a specific gravity of approximately 1.6 gcnf^. \ solutioti of

this nature was obtained by dissolving 1 300 g of zinc

chloride in one litre of distilled water. The minimum ratio

of egg suspension to zinc chloride solution had to be 1:14

(eg. 3 ml. bf egg suspension and 42 ml of zinc chloride

26

A

solution), otherwise it appeared that not all the eggs would

float. If the concentration of eggs was very high it was

better to increase the ratio to 1:28, or even more, in order

to facilitate counting. The zinc chloride/egg suspension

mixture was thoroughly mixed and a sample rapidly transferred

to the counting chamber of; a McMaster slide using a Pasteur

pipette. The eggs were allowed to rise to underneath the

upper surface of the slide and were then counted

microscopically. Usually three McMaster chambers were

counted and an average egg count per chamber used in the

calculations. The McMaster slide is shown in Figure 4, As

the counting chamber is ] cm square and 0.15 cm deep, the

formula for the number of eggs per ml of egg suspension is

given by;

x (y*z) . 100y

15

where x average egg count

y = size of egg suspension sample in ml

z - amount of zinc chloride suspension added in ml

Knowing the percentage embryonation of the eggs, as

determined in section 3.4, the number of embryonated eggs per

ml could be calculated. Thereafter, a solution containing

150 eggs in 0,2 ml could be prepared for administration to

the test animals.

Experimental Design

C, hepatica eggs were administered to the experimental mice

on day one of the experiment (section 3,6.1) and their body

FIGURE 4 McMasfcer

29

weights recorded. On days fourteen to eighteen post

inoculation the drugs were administered to the mice according

to their daily body weights (section 3.6.2). On day twenty-

nine post inoculation each animal was weighed, anaesthetised

with ether, and then its jugular vein was severed for

exsanguination so that the liver did not act as a reservoir

for blood and make the liver weights inconsistent. The body

cavity was opened up, any gross abnormalities noted, and the

liver removed and weighed. The livers from each group of

mice were photographed. Each liver was wrapped individually

in a plastic bag and labelled with the experiment code, mouse

number, cage number, drug and dose-level. The mouse livers

were frozen at -70°G for* a minimum of seventy-two hours in

order to render the C. hepatica eggs uninfective (Wright,

1961). The livers were then processed by means of a modified

liver digestion technique (section 3.6.3). The number of

eggs pet gram of liver was calculated and the results

evaluated (sections 3.6.4 and 3*6.5).

Pilot experiments were first performed with each. drug. This

involved using very high dose-1 evels of the test drug to

determine whether or not the drug had any potential use

against C» hepatica. Three dose-levels were used in each

case, doses at each level being administered to five mice

from days fourteen to eighteen of the infection, A control

group of five infected but untreated mice was used, For

drugs which appeared to bo having an advantageous effect on

the infection, further experiments were performed using much

lower dose-levels *

3.6.1 Infection of Test Animals

The test animals were inoculated per os with

approximately 150 embryonated C. hepatica eggs, by means

of a blunted metal cannula attached to a 1 ml disposable

plastic syringe. The dosing syringe is illustrated in

30

Figure 5 and the dosing procedure in Figures 6a and 6b,

Since the eggs sink rapidly in water, care had to be

taken to shake the bottle containing the eggs before

filling the syringe, in order to administer the required

number of eggs as accurately as possible.

3.6*2 Drug Administration

A list of drugs used, the manufacturers and other

pertinent information is given in Appendix A. Each drug

came in one of the following forms:

(i) Powder soluble in water

(ii) Powder insoluble in water

(iii) Suspension rniscible with water

(iv) Suspension not rniscible with water

A powder which was insoluble in wafer was suspended in a

solution of gum tragacanth by means of a homogeniser. A

suspension not rniscible with water was supplied with a

suitable fluid for dilution^ which was also used as a

placebo for the control animals where applicable. The

drugs were supplied in known concentrations.

Consequently, the required dose-levels could be prepared

so that each drug was administered in an amount of 1 ml

fluid per 100 g body weight. Thus, if the dose-level

was 500 mg/kg, then each 1 ml of the solution to be

administered contained 50 mg of the drug. The drugs

were prepared daily and given to the test animals as in

section 3.6.1, Drugs were, administered in ascending

order of concentration and a new syringe was used for

each drug, the dosing needle having been thoroughly

washed, Control groups were dosed with distilled water,

gum tragacanth or placebo, according to which had been

used for drug administration in the particular

experiment concerned.

FIGURE 5

FIGURE 6a Dosing Procedure ~ I

FIGURE 6b Doainp; Procedure - II

M odified Liver M p.eflfclon Technique

The technique described by Matsusaki (1951) (cited by

Vollerthun et al., 1974; Lcimmler and Grliner, 1976;M ttHM PW«WW>

Zahner et al ., 1976) was modified and used as follows:

A solution containing 0.7 g pepsin, 6 ml normal (molar)

hydrochloric acid and 94 ml distilled water was placed

in a 250 ml round-bottomed flask. The liver was cut

into small pieces and added to the solution, the flask

being clearly labelled. The flask was held in a water-

bath maintained at 37,5°C and stirred by means of a

vibrating stirrer obtained from Chemapec (Pty) Ltd.,

P.O. Box 9051, Johannesburg, South Africa. The

apparatus is shown in Figure 7. The flask was stirred

for a minimum of three hours, or until all the fibrotic,

masses had been broken up. The resulting solution was

made up »:i 100 ml with distilled water and placed in a

sealed flask it 4°C unr.il required for examination.

Egg wivuueinp, ~ for Liver Examination1 *■ • 'UKMMMtWOTM ........................................ ,Hllllim*'W W H I uhl'W HHlM WImi*

Thri iiuspe*-»ion was thoroughly mixed and a 3 ml

K-H.juu i re •! wifeh a graduated pipette. 42 ml of

j .6 g e n f z i n c chloride solution was added to this

sample. This solution was thoroughly mixed and a sample

taken quickly with a Pasteur pipette and run into a

McMaster counting chamber. Three samples were examined

microscopically and the eggs counted is described in

flection 3.5.

The formula for the weight of liver examined per

McMaater chamber (Wm) is given by;

Wtti * V . 3 . 0,15

200 . 45

Wm » W , 5 . 10“5

Where I<? weight of the mouse liver in grams.

?i •. : v!lf eggs per gram of liver (EL) is given by;

v; 1,

- m

*. ■»: ■■■ ;i\fv;rsgc egg count per McMaM::r chamber.

Tti<; percentage ►'eduction in the number of eggs present

in i;he livci is given by:

(Average EL for givci

R e d u c t i o n - 1 0 0 d r u a / d o s e - l e v e l j . 1 0 0

(Average EL for Control group)

The liver weight per 10 g body weight was calculated for

each mouse, and the Dunne11* s test, for comparison of

treatment means with the control, was performed to

compare liver weights between dosed and control groups

(Dunnett, 1964),

A linear regression analysis was utilized to investigate

the relationship between the number of eggs per gram of

liver and the liver weight.

Scanning Electron Microscopy (SEM)

For drugs which proved to be effective, as determined by

the egg counts from the low-dose experiments, groups of

five mice were infected with C. hepatica, as in section

3,6.1, and treated with drugs, as in section 3.6.2, The

dose-levels used were the lowest effective dose-levels

for the appropriate drugs, as determined by the low-dose

experiments. The mice were killed on day nineteen post

inoculation by means of ether, A control group of five

mice was also infected with C. hepatica and dosed with

distilled water.

Livers were removed and some of the worms extracted by

tuicrod Is section, Worms were immediately fixed in

glutaraldehyde at room temperature (to prevent excessive

shrinkage) for a minimum of one hour. The worms were

then washed in cacodylate buffer and post-fixed in

osmium tetroxide at 4°C for at least one hour, or until

the worms had turned black. The were then washed

in buffer and dehydrated at 4°w t'trough an ascending

alcohol series. The wox*ms were critical point dried,

mounted and gold-coated before, examination under the

scanning electron microscope (after Wrigbt, 1978),

RESULTS

Effects of T)rugs

A summary of the high-dose pilot experiment results is

given in Table 3 and the control data in Table 4. Full

experimental data from which these tables are derived

are given in Appendix B, In ordur for the drug to be

deemed effective, it was required to cause more than 99

per cent reduction (against the control) in the number

of eggs present in the livers of the experimental mice*.

The column scoring the liver appearance is, admittedly,

a subjective measurement, but as can be seen from the

photographs which follow, the liver appearance

(including apparent fibrosis, colour and texture) is a

reasonable indication of the degree of infestation with

G. hepatica. In the pilot experiments, four drugs

showed the required degree of efficacy, and these were

then subjected to low-dose trials. They were

albendozole (Valbazen © ), febante'i. (Rintal ($) ),

mebendaz (Vermox (§) ) and oxfendazole (Systanvex ® ).

Also in the case of these drugs, the Dunnett's test to

determine any significant difference between the control

and treated liver weights is recorded as being highly

significant at one per cent, i.e. there is a very

significant difference between the control and treated

liver weights, which has not arisen by chance.

The results of the low-dose experiments are. given in

Table 5 and the control data in Table 6. Full

experimental data from which these tables are derived is

given in Appendix C. The lowest dose-level in these

experiments was chosen so that the total dose

administered fel] within, or was very near to, the dose-

levels recommended by tlu manufacturers.

TABLE 3 Results of Pilot Experiments

DRUG NAMEDOSE

mg/kgX5

NUMBER OF MICE TO SURVIVE

LIVERAPPEARANCE1

EGGS/ g LIVER DUHNETT* S TEST

CONTROLGROUP

x x 104 % REDUCTION

Albendazole 125 5 + 4- 0.1 99,8 0.01 B(Valbazen (R)) 250 5 ++ 0.2 99.7 0.01 B

500 5 ++ 0.3 99,6 0.01 B

Amoscanate 125 5 0 163.1 20,2 N.S. El250 5 0 150.7 26.3 M.S. El500 2 0 255.8 0 H.5. El

Febantal 62,5 5 ++ 0,1 99.9 0.01 C(Rintal (R)) 125 5 ++ 0.4 99,8 0.01 C

250 5 •f + 0,2 99.9 0.01 C

Mebendazole P.25 \ ++ 0,1 99.9 0.01 A1(Vermox(g)) 12.5 5 ++ 0.1 99.9 0,01 At

25 5 ++ 0,1 99.9 0,01 A1

Niclosamide 125 4 0 153.6 9.5 N.S. C(Lintex (§)) 250 4 0 135.8 20.0 N.S. C

500 5 0 67.5 60.2 N.S. C

Oxantniquine 125 5 0 90.3 0 N.S. fcc(Yansil (§)) 250 5 0 74.5 1,3 0.01 E2

500 2 0 73.2 3.1 N.S. E2

Oxfendazolfc ' 56.25 5 | + 0 100 0.01 0(Systaraex (f)) 112.5 5 + 0 100 0.01 0

225 5 + 0 100 0.01 0

Gxyclozanide 85,0 5 0 26.6 65.8 N.S, B(ICI Liver 170 5 0 40.7 47,6 N.S. BFluke Remedy (R)) 340 4 o .... 5.3 93,1 N.S. B

Plperaz-ine 125 5 0 123,7 0 N.S, Badlfijte 250 5 0 124.3 0 N.S. 8(.Ascarad'iria f); SOO 5 0 175,6 0 0.05 B

Piperaz v<te 125 5 0 196,0 28,4 N.S, Acitrate 250 5 0 110.3 59.7 N.S. A( R id®) 500 5 0 248.6 9.1 N.S. A

Piperazine 125 2 0 100.0 0 N,S, Ddihydrochloride 250 4 0 133. v 0 N.S. D(Wazlne (R)) 500 4 0 157.1 0 N.S. D

Praziquantel 50.0 5 0 72,9 73.4 N.S. A(Droncit (§)) 100 b 0 229.7 16.1 N.S. A

200 0 "* ** ** A

Pyrantel 125 5 0 66.7 75.6 N.S. A(Combantrin (R)) 250 5 0 86.5 68.4 N.S. A

500 5 0 117,0 57.3 N.S. A

Rafoxanide 62,5 5 + 66.4 60,9 0,01 C(Ranlde ® ) 125 3 + 70.1 58.7 0.01 C

250 0 ” C

(Tnnt 1nur*d 1

TABLE 3 (Continued) ..........

* Key To Liver Appearance Symbols

0 : No apparent difference in appearance from the control

livers.

+ I Some reduction in fibrosis apparent.

++ ; Considerable reduction in fibrosis.

TABLE 4 Results for Pilot Experiment Control Groups

CONTROL GROUP

NUMBER OP

MICE TO

SURVIVE

x EGGS/g LIVER

x 104

A1 5 120.7

A 5 273.7

B 5 77.6

C 5 169,7

1) . 5 87.9

El 5 204.3

E2 5 75,5

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TABLT 5 (Continued) . . . .

Key To Liver Appearance Symbols

No apparent difference, in appearance from the control

livers.

Some reduction in fibrosis apparent.

Considerable reduction in fibrosis,

0

TABLE 6 Results for Low-Doso Experiment Control Group?.

CONTROL GROUP

NUMBER OF

MICE TO

SURVIVE

x EGGS/g LIVER

X 104

F 5 33.9

H 5 56,8

J 5 66.9

For each drug a trend may be seen, which will be

described below. In order for the experimental,

photographs to be assessed objectively, reference should

be made to Figures 8 and 9 which are photographs of

livers of uninoculatecl and inoculated control mice,

respectively. Bearing in mind that the scales are

different, the uninoeulated mouse livers (Figure 7) are

dark blood red in colour and are generally smaller than

the inoculated controls (Figure 8). The inoculated

control mouse livers are paler, because of the fibrotic

masses of eggs located within the liver parenchyma.

These livers are heavier than normal and firm to the

touch, not soft and easily damaged as is a normal liver.

Albendazole (Valbazen©); Figures ID - 19

The lowest: dose of this drug administered to mice was

1,88 mg/kg (total dose 9.40 mg/kg). At this level the

livers closely resembled the. inoculated control livers

in colour and texture; tbu, Dunne tfc’s test was not

significant and the egg count was reduced by only 20.5

per cent. If Figures 10 - 14 (dose-levels from

,1.88 mg/kg to 30.0 mg/kg) are examined, a progression

indicating an improvement in the condition of the liver

may be seen. The fibrosis is gradually reduced untxi at

30.0 mg/kg there are only a few white fibrotic areas

visible, the livers being predominantly a healthy colour

and more natural in texture, At 30,1) mg/kg the egg

burden is reduced by 99.8 per cent and the Dunnett's

test shows a highly significant difference in weight

between treated and control livers, Figures 15 - 19

(dose-levels from 60.0 mg/kg to 500 mg/kg) show livers

which are almost completely tiealthy, having only a few

small pockets of fibrosis.

Tg

7+3

4.1.2 Febantel (Rint.il ® ) ; Figures 2.0 - 29

For this compound the; lowest dost? administered was 1.25

mg/kg (total dose 6.25 mg/kg). At this level the livers

were identical to those of the inoculated controls in

colour, texture and apparent abundant fibrosis. The

Bunnett's test was not signifant and there was no

reduction in egg count. If one examines Figures 20 - 25

(dose-levels from 1.25 rag/kg to 30.0 mg/kg) a similar

picture of improvement in the liver condition to that

for albendazole, may be seen. There are no visible

differences between Figures; 20, 21 and 22 (1.25 mg/kg,

2.50 mg/kg and 5.00 mg/kg respectively), and in fact

these livers show no numerical, reduction in the number

of eggs present. However, at 10.0 mg/kg (Figure 23),

the fibrosis can be seen to be slightly reduced; and at

15.0 mg/kg and 30,0 mg/kg (Figures 24 and 25) the liver

is seen to be normal in colour (and normal as regards

texture), with only a few pale fibrotic areas visible.

It is at 30.0 mg/kg tb t the egg count is reduced by

more than 99 per cent. ’!;.<> Durmett's test also reflects

these changes, from being 5 per cent significant at

10.0 mg/kg to highly significant (one per cent) at

15.0 mg/kg and 30,0 mg/kg. Figures 26 -* 29 (dose-levels

from 60.0 mg/kg to 250.0 mg/kg) show livers which are

almost normal. Fibrosis is almost entirely absent at

the high dose-1evels.

4.1.3 Mebendazole ( V e r m o x ) ; Figures 30 ~ 33

Mebendazole (Vermox © ) is the most effective

anthelmintic against 0. hepatica investigated to date.

This drug is highly effective against this parasite at

as little as 3,13 mg/kg (total dose 15.63 mg/kg).

Figure 30 (3,13 mg/kg) shows normal-coloured livers with

a few pale fibrotic patches. This picture continues in

Figures 31 - 33 (dose-levels 6.25 mg/lcg to 25.0 mg/kg).

- Y ' \

-

k i? ‘

44

The Dunnett's to.at also shows the difference in the

liver weights (as compared to the controls) to be highly

significant at all the dose-levels used.

4.1.4 Oxfendazole (Sy8tamp* ,(]R) ) ; Figures 34 - 43

The lowest dose of this drug to be administered wag 1.25

mg/kg (total dose 6.25 mg/kg), Figures 34 ~ 37 (dose-

levels from 1.25 mg/kg i,o 10.0 mg/kg) reveal livers

which still have large amounts of fibrosis present»

although in some livers it is reduced, giving rise to a

pale, mottled appearance. At dose-leve.ls oC 5,0 mg/kg

and 10.0 mg/kg there is some reduct if"; in the egg

counts i although the Dimnett's t<-..~ l remains non

significant. Between 10,0 mg/kg and 17..5 mg/'ig ^Figures

37 and 38) there is a dramatic change. At 12.5 mg/kg

the livers have a more normal blood-red colour and

fibrosis is greatly reduced. The number of eggs present

is reduced by 99.8 per cent at 12.5 mg/kgj and the

Dunned;1 e test shows a highly signif Lei at difference

between the control and experimental liver ’/‘j.hts. The

liver appearance continues to improve > the higher

dose-1 eve Is (Figures 39 ~ 43; dose-le,, ' i.B ire •• 25

mg/kg to 225 mg/kg), reveal ing livers c f normal colour

and siae, and having only a few pale fibrofic patches.

4.1*5 Other Drugs Tested ; Figures 44 - 53

Figures 44 - 53 show the livers oE mice from the hifbest

dose groups of all the remaining drugs tested. None of

these drugs caused any significant reduction in egg

numbers. The photographs show livers which are similar

in appearance (and texture) to the inoculated control

mouse livers shown in Figure 9.

I

V.

FIGURE

FIGURE

45

MOUSE, no 380CAGE no 34 Uninoculated

Control

M O U S E no 381 CAGE no 34 Uninoculated

Control

mM O U S E no 382 CAGE no 34 Uninoculated

Control

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Uninoculatod Control

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Livers from Uni nocul aU'>d Control Mice

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FIGURE 11 Livers from Miee Dosod with 3.75 mg/kR Albcndazole

(ValbaKon (® )

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(Rintal (JO )

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FIGURE

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«pt cM O U SE no JOS

CAtiE no 28

0 2 5 - ;>it j fjtfJTAJ,

EiCPT £•MOUSE no 18? CAGE no 28sa.r g/kn mini

m

i m rMOUSL no 500 CAGE no 23 « J SKfl/K® ft fiTAI.

cxpr tM O U ,;i: no -i09

CAOu no 2S*? 3)Vjj/!(j} JUNTAS,

FIGURE 27 Livers from Mice Dosed with 62.5 mg/kg Febantel

(Rintal © )

Scale bar indicates 25 mm i__._„j

FIGURE 2

FIGURE

ewt e MOUi.fc n-j CAW ntf 2J

WSrs/kr, P.WTAt

m t - cMGIK'* ro V>? CAP i no «•*

t t? " i-i." Sl'iTSC.

t’XI’T CMOl'S. 1’34CACB l lw

IP S r-v „! fttNTRt

£XPf C

MDUSl' no P2 CAD1’' no »5r5a/k3 RtKTAI,

em- cMOUf/f no 63 CAf nr- <.9

(USTKt

Livers from Mice Dosed with 125 mg/kg Febantel

(ftintal ® )

Scale bar indicates 25 mm t— ___ i

em cMOUSE »m> tS5 CASE no 30a s f i» 3 « c i iv:nr<\,

'30EXPt CHOUSE n« CACfl tn. JOzsbtpjm n"‘fai

z m cMousr. no m ’CaCE ''no 30 tsdm^s mum,

s m ewmjst no too CA&i n« Ji 2B8B9/H0 H'KTM.

CXi’T cM 0 ‘J f E r»o ^ 9 C A tiv ! n o .sii,S50mo/ha R'tStSi,

9 Livers from M ice Dosed with 250 mg/kg Febantel

(Rintal ® )


FIGURE

FIGURE

EXPT J H O U S E no CAGE no 2

a.ia rce /ito vesm ox

expt jM OUSE no 561 CAGE no 2

J . ia s ta /k g VERMOX

EXPT JM O U S E no 562 CAGE y\ o 2 3,S3«g/f!0 VERMOX

EXPT JM O U S E no 563CAGE no 2S .t t ir rn /k G VERMOX

EXPT J


3. tSmfl/fcg VERMOX

30 Livers from Mice Dosed with 3.13 tng/kg Mebendazole

(Vermox (R))

Scale bar indicates 25 ran

E'/n jM O U S E no S65 CAGE no 3

G .S S n ig A g VF.RMOX

EXPT J

M O U S E no S66 CAGE no 3

6 .S S m g/l<o VFRMOX

EXPT J

M OUSE no 508 CAGE no 3 G. 25 mg/(to VPRMOX

EXPT J

M O U S E no 567 CAGE no 3

6 . S $ ) h , / k a VERMOX

EXPT d

MOUSE no 569 CAGE no 3G .S S m g/kg VERMOX

31 Livers front Mice Doaed with 6.25 mg/lcg Mebendazole

(Vermox (R) )


EXPT JM OUSE no 570 CACi no 4

12.Sia.jAg VERMOX

e x p t j

MOUSE no 571 CAOti no 4

1S ,S m §/ftg VERMOX

EXPT J

M O U S E no 572 C/-U.V:;. no A12 ,B m g /k g VERMOX

EXPT J EXPT J

M OUSE no 573 M O U S E no 574CAGli no 4 4,'AUi- BO 4■ 1 C .5 r*g /k g VERM 0X Ig .S n r j / k g vCRMOX

FIGURE 32 Livex's from Mice D o s e d with 12,5 mg/kR Mebendazole

(Vetmox ® )

Scale, bar indicates 25 mra

M I C E D O S E O W I T H

2 5 m g / k g V E R M O X

FIGURE 33 Livers from M ice. Dosed m th 25,0 wg/tcp; Mebendazole

(Vermox ® )x i n M n n im w u nn w m.ti uitmmt

Scale bar indicates 25 mm i_____________j

FIGURE

FIGURE

: m ' rno 335 oo 7

1JSmg/Vj 'm?AMEi{

FXiM >•

MOUSE ho 336

C A (ili f,o 7

1U5w3'&3 SWAMiX

u x p t r

MOUSE no 337C AGS. rt & ? «s«g/»} CYSTfiW'X

£xrr r

no 336vAt?t, r-a 7 t<!&mx'ti„ .K’.fA M

{X P T F ' '

MOUSf. no 339

CA vie no ?1 ii’jw.'l/k? is fViAMf*

34 Livers from Hiee Dosed with 1,25 mg/kg ftxfendazole

(Systamex © )

Scale bar indicates 25 mm I-----

m t fMOlfSl- no 340

CAQVi fta 8g.5»|J/!<9 'j.ViJAMf X.

exer fMOUSE ny 341

CAi»E no 8

? F>mn >i<3 CiVjTftMex

C m i-MOUSE my 342 CAGE ro 5£ . U n j / l y S V ? JAM ES ’

EXPT IMOUSE no 343

UAOt. no 8

ILSin^ft.) StSfAMEX

pxpr rMC1USE no 344

CAGii no fl

srsTAMrx

35 Livers £rom Mice Dosed with 2.50 mg/kg Oxfendazole

(Systamex (§) )

Scale bar indicates 25 mm j— ----

FIGURE

FIGURE

n u n FMOUSE rto 345 CAGE no 9 Jtea,*t<_5 Sv'TAMtX

! Xl't !■

MOUSE tin m CAGC-: no 0

5 ,0 m m •>'i'.XAhXC'K

« m fM Q U S t no 24S ‘

CASE na 3

O T ? rMOUSt fit1 3A7 CA6E M 3 3 Ofsg/k-3 57SrAM£)f

i'XPt FMOU&i" fift 3.V,j CAGfe. i.*. a

36 Livers from Mice Hosed with 5<00 roR/kg 0y'cmdazolo

(S y s tn m D X (R) )w <m W * n'» « " » ' i«' i*«aau u«


fxpr fM0U51- na 350 CAUt ro Yi «) ntua/k'} 'try>mu

iffX !M O U SF. n o 3 6 ' CA&fc. f*o 13

'iY-STAtffx

i’jfVT i‘MOUw: no 352

CACe no 13

ni n(.:i 'k.: ;>0!Ai'PX

c m fMOUSE no 383

CAGE no n 10 Oraj/St!) 3Vr,TAME)f

KXPT FMOUSE no 354 CAiSt.-. no 13

1&0m8,'IQ HSrAMEX

37 Livers from Mice Dosed with 10.0 mg/kg Oxfendazolew > iwm ii » i i ^ j w iu m i w Biii«i<wn»t*m w i»>— *»'*<*» i»i«»w «»» im i inH'.iiwwif ■ni » w»»t» »*jr|w>w—wMMM.MKJMWwwjrmiivw

(Syatamex (f§ )


m>r iiMOUSE no 470 CAGfci no 6VI S ng/fe fl SYSTAMEX.

EXPT H

MOUSE no 471 CACifi no 8 .12 5 snortS 3 VST AMEX

KPT HMOUSE no 473 CAGL -no 6

la.StPO/kg SY5TAMEX

EXPT H

MOUSE no 472 ■CAOS; no G12.5*na/le£( SYSTAMEX

EXPT H

MOUSE no 474 CAOt. no 6 .. 12,S « g /k g S ifSTAM E*

FIGURE 38 Livers from Mice Dosed with 12-5 rag/kg Oxfendazole

(Systamex <$£} )

Scale bar indicates 25 mm h -i

r m ii

MOUSC no 475 CA©£ no 7

SYSTAMKK

EXPT H

M O U S E no 47S

CAGE no 7 .2S.0fr3/((g SYSTAMEX

4 f | i

EXPT H c

MOUSE no 477 CAGE no 7 85 Smg/fcu 5V8TfiMEX

EXPT Ii

MOUSH no 478 CAGii no 7ZS.Owti/kn SYSTAMEJi

EXPT H

MOUSE no 479 CAGE no 7as.a !r,0 /f«g SYSTAMEJf

FIGURE 39 Livers from Mice Dosed with 25.0 mg/kg Oxfendazole

(SvstamexCR) )


EXP7 H


SCi.Oreg/^ S Y S T f t M E X

EXPT H

MOUSE no 481CAGC no 8 :SO.0RR/f.g 8YSTAMEX:

EXPT H

;MOUSE no 482 ;CA&E no 8 HJO.Ons/fe" SI'S I A M E X

£XPT i! i.:

MOUSE no 483 CAGK no 8

KXPT ?i


i fa .Oirg ,kg SVS'TfiMcX

FIGURE 40 Livers from Mice Dosed with 50*0 mg/kg Oxfendazole

(Systamex >%) )

Scale bar indicates 25 mm i_______ i

KX»T 8MUUEi* pd 224

C A &a no 30

80.fc$K3/1iSSf9TAM£<

m"-i aM O U SE no 225

CAGu no 38

M,?£prt/!!2S5fST,lM2X

6 W & " M O U S E no 226

CAGfc! no 3B

$8,2$X3/kg$Y$TAM£X

ESf»T 0MOiiSs: no 22?

CAOii nu jJ) 8'!,20n>i)/t(fi5Y5UMFX

EXfT 0

MOUSE no '. 'IS CAOk, no 38

BB-SSlrs/kBSYSTAMEX

FIGURE 41 Livers £rom Mice Dosed with 56.25 mg/kg Oxfendazole

(Systamex © )

Scale bar indicates 25 mm j- - - - - - J

62

FIGURE 42

F IG U R E 43

%E X P T 0MOUSE m 223 CAOE no 33'-1 112. EM3/ IsafS V5TAME*

sx t-T r» ■ -: M O U S £ n o 2 3 0 CAfit no ,19

w.sw/wsYsr/imx

EXPT 0 /",MOUSE '■ no 231 CAGE no 33112,Str!)/kB5YSTftMEX

m i e*M(V:-5 '~ no 232

CAGE no 35

VEXJ't

r-o 233 CA«SE n o 33

Tfg.Srrjf/fcjSYSIAMEX

Livers from Mice P osed with 112.5 mg/kg Qxfendazole

(Systamex(R) )


apt 0 'MOUSE no m CAGE no 40 SSSffa/k-JiYSMMEX

Exf't r -;;,,M fo .iw rtc 235 oAtec fiQ "*40.< t:ps-j/k(jSyiTfl«EX

EXP5 0 f ' " ‘ ■ MOUSE r CAGE jK , , g?si>j®sJ|rerVTii£i

rot 0M OU , no 200

C A G t no (\ZSS'itrfl/kfjSYaTAMEX

EXPT 0

MOUSE no 202

CAGE no 435iS&ns8/ita5YSTAMt‘X

Livers from Mice Dosed wi.:h 225 mg/kg Qxfendazole

( Systatnex ® )

Scale bar indicates 25 mm 1™--- -— I

ji*-

EXf'T £

MOUSE no 263 CAGE no 3 SOQmB^sAMOSCABAtt

EXPT E ,

MOUSE no 204 CA^iE no 3

500wg/t3AM0SCANAt£

FIGURE 44 Livers from Hie "d with 500 mg/kg Amoscanat e

Scale bar indicates 25 mm |______ |

v(pt e-mouse na'se CAGE aa 28 soBr.'jAa tiitra-

f m rM O U f i P r. , 1 e ?

t\i t. r.fj 28 C-raJkj iffitEX

exft cM f'U f.f* n o C8

C.A,- , n o 26

iGSwj-Jt.} ; <KTEX

sm cMOUSE no OS

CA0E no 2660Cnr,fl/fig Mf.TCX

tXFt CMOU5K .no to CA>3E no 26 fifKWHt unrex

FIGURE 45 Livers from Mico Dosed with 500 mg/kg Niclosamide

(Lintex ® )

Scale bar indicates 25 mm I------1

FIGURE 46

FIGURE 47

expt eMOUSE no 285 CAGE no a 500«g /ks VANSil

EXPT £

MOUSE no 287 CAGE no 8 SQOmg/'kg VANSft

Livers from Mice Dosed with 500 mg/kg Oxamniqulne

(Vansil® )

Scale bar indicates 25 mm |_____

txpi nr-’iOUGi-: (H, no CAOH r»o 13

i/:r. ii,i«

£ xp t n MOUSt: no tit

fio 13-MtKn/^UiHcrfMa

C xp t 0 MuUSE no tt2UAGSi no f3 34flrfii/l'clG i.}.i-rfl*k»

Expt ft MOlT,i: no 114

n o 13

340<’"'>, t> I . ' i.-m ! lufco

Livers from Mice Dosed with 340 mg/kg Oxyclozanide

(ICI, Liver Fluke Remedy ® )

Scale bar indicates 25 mm l__ __j

■fci. w w

■ - .m

I

k

65

t

E xp t f>

MOOSE, ho 145 CAGE no ?.0 SU0ma?H AiCAAuiJlhft

ft xp t lhM OUSE n o 148 C A 6 . . n o iiO

Se^iiiiVt!) A ' "A.uaiff.’t

E xp i 0M O U S E n o m CAf.'ii' no 20 WOmjf'itg AS ARAOiNfl

E xp t B M O v iftH fsa 140

CAfeC no 20 sOQ’t-a/w-3 Ar-v?wmA

G.xpl B MOUSl- v.o 149 CAUii no 20 asoi-(V.',nfi8WB

FIGURE 48 Livers from Mice Dosed with 500 raft/kg Piperazine

Adepate (Ascaradina f§) )


gjtef a

M O U S E a o 6 S

CmSE no «SOOirQ/fcs R,l

Expt A M0U3G O') 86 CASfc no a'■ocmg/Kj a,6

E x p t A

MOUl'ii r.'j 0? W » 8 E n o a

^ wth} no

E xp f AMOUSE r,a 88 ('■ -Ofc no u

BSOirj/lij !;i'f

E x p t A

M O U SE h o 89

C \6!- i) o ‘i 50‘- u.-n/hq U ;j

FIGURE 49 Livers from Micc Dosed with 500 mg/kg Piperazine

Citrate (Rid ® )

Scale bar indicates 25 mm }_____ [

# *» f_ _* ■AwwwjX.

EXPT 0MOUSE no 2 U CAGE no 34 SOOmo/xg WfiiiKfc*

expr tf■MOUSE no 356CAGE no 35 'so&raj/ku ivazmtr

EXPT u ■MOUSE no 215 CAGE no 34

.SOOmg/kj v /A tM

■EXPT S)MOUSii no 217 CAGE no 35 SOOfliS/Iiff WASHNE

FIGURE SO Livers from Mice Dosed with 500 mp,/kg Piperaziae

Dihydrochloride (Waaino ® )

Scale bar indicates 25 mm [- i

Hxpt A MOUftt ft a OAO:. no 5 lOtimff/tij WtCilClT

0F, *pt A MOUSF «d VI CAf.i?. 5io 5ttV' vk«) uRPyCrt

Exp I A MOUSE no 72 CA&. no 8 ii)t» hi/*.? cnc?;>'t'f

Expt A M'JUSli no 73i * \ no 8 100 « ,'kD tB-KlT

Expt A MOUSE no 74 CAGE no S m m m tsROfjiit

FIGURE 51. Livers from Mice Dosed with 100 mg/kg Praziquantel

(Droncit (R) )

Scale bai: indicates 25 ram |______j

Author Cheetham R FName of thesis The effects of drugs on experimental hepatic capillariasis in mice 1984

PUBLISHER:University of the Witwatersrand, Johannesburg

©2013

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