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)
Scanning Electron Micrograph of
C. hepatica Eggs (x 2 000)
Scanning Electron Micrograph of
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
Albendazole (Valbazen © )
Livers from Mice Dosed with 15.0 mg/kg
Albendazole (Valbazen © )
Livers from Mice Dosed with 30,0 mg/kg
Albendazole (Valbaaen © )
Livers from Mice Dosed with 60.0 rag/kg
Albendazole (Valbazen © )
Livers from Mice Dosed with 120 mg/lcg
Albendazole (Valbazen © )
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 © )
Livers from Mice Dosed with 500 mg/kg
Albendazole (Valbazen ® )
Livers Erom Mice Dosed with 1.25 mg/kg
Febantel (Rinfcal (g) )
Livers from Mice Dosed with 2.50 mg/kg
Febantel (Rintal. CR) )
Livers from Mice Dosed with 5.00 mg/kg
Febantel (Rintal © )
Livers from Mice Dosed with 10.0 mg/kg
Febantel (Rintal ($) )
Livers from Mice Dosed with 15.0 mg/kg
Febantel (Rintal )
Livers from Mice Dosed with 30.0 mg/kg
Febantel (Rintal )
Livers from Mice Dosed with 60.0 mg/kg
Febantel (Rintal (§))
Livers from Mice Dosed with 62,5 mg/kg
Febantel (Rintal ® )
Livers from Mice Dosed with 125 mg/kg
Febantel (Rintal (g) )
Livers from Mice Dosed with 250 mg/kg
Febantel (Rintal (§) )
Livers from Mice Dosed with 3.13 mg/kg
Mebendazole (Vermox 0 )
Livers from Mice Dosed with 6.25 mg/kg
Mebendazole (Vermox © )
Livers from Mice Dosed with 12,5 mg/kg
Mebendazole (Vermox © )
Livers from Mice Dosed with 25.0 mg/kg
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
Livers from Mice Dosed with 1.25 mg/kg
Oxfendazole (Systamex©)
Livers from Mice Dosed with 2,50 rag/kg
Oxfendaziole (Systamex © )
Livers from Mice Dosed with 5.00 mg/kg
Oxfendazole (Systamex © )
Livers from Mice Dosed with 10,0 mg/kg
Qxfendazole ( Systamex©)
Livers from Mice Dosed with 12.5 mg/kg
Oxfendazole (Systamex © )
livers from Mice Dosed with 25.0 mg/kg
Oxfendazole (Systamex © )
Livers from Mice Dosed with 50,0 mg/kg
Oxfendazole (Systamex®)
Livesrs from Mice Dosed with 56.25 mg/kg
Oxfendazole ( Systamex©)
Livers from Mice Dosed with 112.5 mg/kg
Oxfendazole (Systamex © )
Livers from Mice Dosed with 225 mg/kg
Offendaxole (Systamex © )
Livei‘8 from Mice Dosed with 500 mg/kg
Amoscanate
Livers from Mice Dosed with 500 mg/kg
Niclosamide (Lintex © )
Livers from Mice Dosed with 500 mg/kg
Oxamniquine (Vansil © )
Livers from Mice Dosed with 340 mg/kg
Oxyclozanide (ICI Liver Fluke Remedy © )
Livers from Mice Dosed with 500 mg/kg
Pip'razine Adepate (Ascaradina © )
Livers from Mice Dosed with 500 "mg/kg
Piperflzine Citrate (Rid © )
Livers from Mice Dosed with 500 mg/kg
Piperazine Dihydrochloride (Wazine © )
Livers from Mice Dosed with 100 mg/kg
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
Worm from a Mouse Dosed with 12.5 mg/kg
Oxfendazole (Systamex © ) (x 1 000) 71
FIGURE 58 Scanning Electron Micrograph of a C. hepatica
Worm from a Mouse Dosed with 30.0 mg/kg
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 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
i l
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41
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
#M O U S E no 383 CAGE no M
Uninoculatod Control
MOUSE'-no 384 CASE no 34
U n inecu lt ted Control
Livers from Uni nocul aU'>d Control Mice
(Note dark blood red colour)
Scale Bar indicates 25 tnm j........|
« & ■fXPT f
MOUSf' mi 3*18
CAtk-. n o K
• cowri
fXPT f
Mousr. ft0 ane »;Avi-- no !.(
rxpT tno 3!>a
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FXffl FM O U t t n n 3 » 7
CAfcik ft o U
CtH'RiK
fiM'T FMOUSii no 359ftAGfi n <■ 14
twmof
Livers from Control Mice Infected with C. hrpatica
(Note paler colour due. to fibrotic egg masses
within liver parenchyma, and increased size)
Scale bar indicates 25 mra
------------------ - 14 — *---- --------------- ................................................ ........ . .^ mnj
F-Xf'r FMOUSE n o 360 CAGE noV namg/Sty V/W-MUiM
LXPT rM O U S t : n o 3 6 1
C A G E n o IS
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nxfn fM O U &U no 3G2
CAttt no IS
i iitivmts
KPT iMOUfjL. . - *t>4 CAGE; no 5lJ
1 S'3fn3/»> r v.U*<>Vt' (V
FIGURE 10 Livers from Mice Dosed with 1.88 rog/lcg Albendazole
(Valbaatm (§) )
Scale bar indicates 25 trim t----,— »
KPT rM O U 1)!" no 38S
: Ki Hi 'i vMfi«kft
f XPT FMOtftib no 366
tvo t01 tix-'nm V*t<n?!K
G*Pf Fm o u s e n o 3 & 7
CA&b no W9 7S<»n/k‘i V.VMiH
JsXPt f
MOUSE no 388 CASE' n© 16 a. tOWQ/hg MM&m
n n fMOUSE-; no 3£*9 CASE no HI•1 VS)MAM
FIGURE 11 Livers from Miee Dosod with 3.75 mg/kR Albcndazole
(ValbaKon (® )
Scale bar indicates 25 mm
m n t-MOUSE no 370 CA&B no 1? Vimvttf) vftitiAftji
EXPT f
MOUSE no 371 CAGE no 1?
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exfr rMOUSf*.no y nCfVot ho *7
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7 r.mg/iig V/il UMfN
f X f f fMOUSE' r * 374
CAGE rtf) 17f Ml-jr/ki) fititiWH
FIGURE 12 Livers from Mice* Dosed with 7.50 mg/kg Albendazole?
(ValbaKen @ )
Scftle bar indicates 25 mm
iwr fMGU5E, no 3?5 C A b ii no ■«<5 0raykrj Vfti.WUHt
r,m (W0USC »o 376 LAUi. >io 18
*R nfnj.'k,) ¥4tMKH'
fAM *MOUSE no 377 CA6t% no 58
iPa»3f!t,j vmsmw
p m fno 375
CAw no 18 »*■ ’m/kg vM.fmjKH
FIGURE 13 Livers Crow Mice Dofiod wi th 15.0 mg/kg Albendnaole
(Vfllbngon d0 )
Scale bar indicates 25 mm i__„__ i
t XIT f
MOUSE no 379 CASE no m is ftmfl/kfr VAt P.WHN
- - .a&. > amS. t f" " * * * * " 1 1 ' '■■iimrtmi .muLlm
EXPT H
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♦c m h
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EXf-T H
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I<B8S-SXfT M
M O U S E fro 433 CAdSE ny 133’i.Srftfkg
exp: HMOL'f-E no 434 CAGU no ?3 3 3 O fn « /> i l? ALBA JEW
•FIGURE 14 Livers from M ice Dose^ with 30.0 mg/kg Albendazole
(Valbaaen ® )
Scale bar indicates 25 mm
EXPT H
MOUSE no 485 C A Q & n o 14
VftlBAJES
^ Hw a w o i n n E C ^ ■ -
i'XP T H
MOUSE no 496 CAGE no 14f;ft 6trJj/kq VALCAZHft
£S?T H MOUSE noCAGE fuff so Qfr.ffsk. wu
49714
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EXP! H
M O U S E no 4-as CAGh no 14
fiO.Omg/kR MLMIftf
FIGURE 15 Livers from Mice Dosed with 60.0 mg/kg Albendazolem m w iiiw i m u i i h f W p — ww>ii»»wi'u»i>»n«n»»ii'» ^ h»iiaiui*ii«»ii»M»n»«iMUT»«»f m m ^w.u iW M iW i . ininwp in — M m iniiK wiM— «wn'w*iaiPMiinii"iPu iiW i i i-
(Valbazen (§) )
Scale bar indicates 25 mm i .,, t
■r
49
E X P T H
MOUSEQ u* mowg/l-
nono
500
15
f-^ye
w s ' 9
EXPT H
M OUSC no SOI CA&b no IB
EXPT H
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E X P T K
M O U S E no 502 CAyU. no ?S S20 pir-fj/hg i'SUsAZEW
SXPT H
MOUP!? no 5041:
t *
FIGURE 16 Livers from Mice Dosed with 120 mg/kg Albendazole
(Valbazen © )
Scale bar indicates 25 mm j--------j
ftjfpl HM O U S E 116
C A G fi i(D i 4
f?.5i*3/ fr7 Vfil 5f i f r «
KXpfc 0Mounr no m
• noiSStTiYi,1 ‘.f: :vr«
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t, Xp Im o *'- ' m
■?'• ti j ! 4'<n;' /t«; i.r.Tfj
FIGURE 17 Livers from Mice Dosed with 125 mg/kg Albendazole
(Valbazen © )n iy «»iiiiii«»iiTi-iii li— i it ~t~i—*rff ‘ ~r-- .
Scale bar indicates 25 mm t___ _____|
E*pt 6 MOUSE no *20 CAGE no 152 5 0 m g / k g V A L M I E N .
E})pt f>Mt)US£ r<o 121 CAOE no 15 asfttr&'I-g VAL8A2EN
Expt B MOUSE no 122 CAC>£ no JS jlSVK /kff VMMMtH
Exp i R MDUSi.- no CAwE no
123'IS
c w v T r y r t ^ i ‘V T H w n T i ^ r r ”
Expt B MOUSE no 124 CASE no f5£wfc§jr*fj ‘rnVBhZ k?
FIGURE 18Livers from Mice Dosed with 250 mg/kg Albendazole^
(Valbaaen © )
Scale bar indicates 25 mm |~---— •|
Expt B M O U S E no 125
C A G £ n 0 4 6
SOSma/ S VAU8AIEH
E x p t B
MOUSi-; no 126 CAGE ,.no 16
iSOO&'S/Jtfl
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L:
Expt B .MOUSE no >20 CASE '- no 16 £00mg/Kg VALBAZ5N
Expt G MOUS,:. no 129 CAGE no 16 500mo/ti8 VAl.8A7.EN
FIGURE 19Livers from Mice Posed with 500 mg/kg _Albendazole,
(Valbazen (TQ )
Scale bar indicates 25 mm j— :---
FIGURE
expr p
MOUSE no 310 CAGE no ! VSSm8«g RitffAt
EXfr fM O U S E no 311 case no i-1,?SiHtjk,5 QMTAL
expt r
M O U S E no 313
CAGE no 51P5mg/kg m n t
£XPf F
Mouse nq 312 gagr no jl J S p q / l i g M K T A L
m > r f
M OU SE no 314
CAGE no 1
igsmj/k5 JtfNTAJ.
20 Livers from Mice Dosed with 1,25 mp,/kg Febaittel
(Rintal (§) )
Scale bar indicates 25 mm ■i
exn fM O U SE no 315 CAGE, no 2
z.twm fKNWt,
fXCT fM OUSK no 356
CAGE n 0 2 2 itmij/Ku rtmui
EXPT f
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e m iMOUSE no 358 UACt no 2 S.Sng/Rg f l t t l l A l
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FIGURE 21 Livers from Mice Rosed with 2.50 mg/kg Febantelw » » H i . i i . iw lw m »»M W ...........................................................................................................................................................................iuin» <wiun » w .vef^tTW«.»j
(Rintal (JO )
Scale bar indicates 25 mm j---- -— |
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52
h.u-r f- •MOOsr. no 325
ij'AGt no 4 5.QmS/*5 RffiTAi,
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E K F T F
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FIGURE 22 Livers from Mice Posed with 5.00 mg/kg Febantel
(Rintal <g) )
Scale bar indicates 25 mm j______ {
e x p t r
MOUSE no 330 CAGE no S to.OttaAa ri'{?.u
f '' ' M O U S E ' n o 3 3 ! CAGE no 6 la.Owg/Hf! swtfti.
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CXPt f
MOUSE no 332 CAGE no 8 10.0rnma SWAl
■i’XPT F
MOUSE no 334 CAOtt no 6 W.Omg/itff (mui.
FIGURE 23 Livers from Mice Dosed with 10.0 mg/kp; Febantel
(Rintal (K) )
Scale bar indicates 2S mm
FIGURE
FIGURE
EXPT H
MOUSE no 455 CAGE no 5
15 Ofog/ksj R l W T A L
EXPT !!
MOUSE no 458 CASE no 1
IS.Omg/kg f ^ T A L
£Xi>r H
MOUSE no 45? CAGE no 1ISSnnr./kq 9!li7At
EXPTHMOUgE fio 450CAGE no 1
€KPT HMOUSE no 459 CAGE no 1 iQ.Omg/fig fitiVTAL
24 Livers from Mica Dosed wi trh 15.0 mg/kg Febantel
(Rintal (g) )
Scale bar indicates 25 mm
exer h • MOUSE no 460CAGE no 2• SCMJwo/lcsffiNTAt
&cpt h
MOUSE no 461 CAGK no 2 ; aO.OittiJ/fcgRlNTAt,
EXPT H
MOUSE no 462.Cage no 2'• SaOrsg/^m^TA!,
HXPT H
MOUSE no 453 CAGE no 2
30.0mg/k>j RfNTAl.
E X P T >1
MOUSE no 464 CAGE no 2
30.0ir.a/Sffl«l»ITAL
25 Livers from Mice Dosod with 30.0 mg/kg Febantcl
(RinCal (J5 )
Scale bar indicates 25 mm |--- ---- J
EXPT H
MOUSE no 465 C AGS no 4
BO.Ou-g/VtgRINrAl
I W HMOUSE na 466 CASK no 4CO«ff0rtgl?mtAL
EXPT H
MOUSE no 467 CABi£ no 4
e&0*0/kSffr'N<At
h ' MOUSE no 460 CAGifc, oo 450.0rr(}/k;t RiKTAS.
CXPT nMOUSE no 469 CAS1-' no A
SO.Omg/kgRIKTM
FIGURE 26 Livers from Mice Doflfid with 60.0 mg/kg Febantel
(Rintal($ )
Scale bar indicates 25 mm |-----'— j
’ EXPT C
MOUS'i ^ W-
CAS£ no W
«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 ® )
Scale bar indicates 25 mm
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
MOUSE no 564 CAGE no 2
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) )
Scale bar indicates 25 mm
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«
Scale bar indicates 25 mm
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§ )
Scale bar indicates 25 mm
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 <$£} )
Scale bar indicates 25 mm h -i
r m ii
MOUSC no 475 CA©£ no 7
SYSTAMKK
EXPT H
M O U S E no 47S
CAGE no 7 .2S.0fr3/((g SYSTAMEX
4 f | i
EXPT H c
MOUSE no 477 CAGE no 7 85 Smg/fcu 5V8TfiMEX
EXPT Ii
MOUSH no 478 CAGii no 7ZS.Owti/kn SYSTAMEJi
EXPT H
MOUSE no 479 CAGE no 7as.a !r,0 /f«g SYSTAMEJf
FIGURE 39 Livers from Mice Dosed with 25.0 mg/kg Oxfendazole
(SvstamexCR) )
Scale bar indicates 25 mm
EXP7 H
MOUSE no 480 CAGE no 8
SCi.Oreg/^ S Y S T f t M E X
EXPT H
MOUSE no 481CAGC no 8 :SO.0RR/f.g 8YSTAMEX:
EXPT H
;MOUSE no 482 ;CA&E no 8 HJO.Ons/fe" SI'S I A M E X
£XPT i! i.:
MOUSE no 483 CAGK no 8
KXPT ?i
MOUSE no 484 CAGE no 8
i fa .Oirg ,kg SVS'TfiMcX
FIGURE 40 Livers from Mice Dosed with 50*0 mg/kg Oxfendazole
(Systamex >%) )
Scale bar indicates 25 mm i_______ i
KX»T 8MUUEi* pd 224
C A &a no 30
80.fc$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) )
Scale bar indicates 25 mm
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§) )
Scale bar indicates 25 mm
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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