Clinical Changes Caused by the Liver Fluke Metorchis conjunctus in

Veterinary Pathology
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Clinical Changes Caused by the Liver Fluke Metorchis conjunctus in Cats
T. G. Watson and N. A. Croll
Vet Pathol 1981 18: 778
DOI: 10.1177/030098588101800608
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Vet. Pathol. 18: 778-785 (1981)
Clinical Changes Caused by the Liver Fluke Metorchis conjunctus in
Cats
T. G. WATSON and N. A. CROLL
Institute of Parasitology, Macdonald College of McGill University, Ste. Anne de Bellevue,
Quebec, Canada
Abstract. Cats infected with metacercariae of the fluke Metorchis conjunctus were followed
clinically through their infection. Cats given 200 metacercariae showed few symptoms. All the
cats passed ova on the 17th day. Three hundred metacercariae caused diarrhea, icterus,
discolored urine, green feces and eosinophilia after 18 to 21 days. Eosinophilia, Ieucine
aminopeptidase and alanine aminotransferase were elevated and remain the best indicators
for metorchiasis. The hematological and serological abnormalities resolved rapidly and were
absent from cats with chronic infection.
Acute lesions (

Metorchis in Cats 119
Materials and Methods
Male and female mixed-breed cats weighing 1.0 to 3.5 kg were treated with piperazine to
expel Toxocara cati and then housed in cages with a mesh bottom and a plate to rest on.
Water was provided ad libitum and each cat had about 100 g of Purina cat chow (Centre
Agricole St. Clet, St. Clet, County Soulanges, P.Q.) daily; feces were examined for ova with
the formalin-ether technique [ 171.
Viable metacercariae were digested from the muscles of the common sucker (Catastomus
commersoni) in artificial gastric medium. They were placed on the back of the cat’s tongue
and were swallowed or flushed down the throat; cats were observed carefully to rule out
vomiting of the inoculum.
Blood collection was done between 0800 and 1000 hours. Cats were sedated with ketamine
hydrochloride (0.05 mg/kg body weight); 4 ml of whole blood was removed by cardiac
puncture with an 18 gauge needle and 5 ml syringe. Heparinked, microhematocrit capillary
tubes were filled with whole blood for leukocyte and eosinophil counts in Unopette diluting
chambers (Becton-Dickinson, Rutherford, N.J.). The hematocrit packed cell volume was
measured from the remaining capillary. The remaining blood was clotted at 22°C for 30 to 40
minutes. The serum was frozen at - 17°C in 2- or 4-dram vials. The following tests were done:
total serum protein, albumin, and globulin (Sigma, St. Louis, Mo.), with a Coleman Junior I1
Model 6/20 spectrophotometer (Canadian Coleman Co. Ltd., Toronto, Ont.); serum protein
electrophoresis was done with a Corning 740 densitometer; serum bilirubin, leucine amino-
peptidase and alanine aminotransferase activity were determined by Boehringer Ingelheim
Mannheim GmbH Diagnostica (Waterford, Conn.); alkaline phosphatase was quantified by
Sigma (St. Louis, Mo.) technical procedures.
Three experimental infections were given as follows: Experiment I: Five cats, coded 1 to 5,
were given 200 metacercariae apiece while two controls were given placebo. Blood samples
were taken two days prior to infection, immediately prior to infection and on days 2, 4, 6, 8,
12,20 and 32 post-infection, when the cats were killed. Experiment 2: Cats 6 and 7 each were
given 300 metacercariae. Blood was sampled before infection and at weekly intervals after
infection until they were killed at day 59 (cat 6) and day 78 (cat 7). Experiment 3: Cats 8 to 12,
three female and two male littermates weighing 1.5 to 3.0 kg, were used. Two cats received 200
metacercariae, two received 300 and one was given placebo. Blood was collected before
infection and on days 7, 14, 17, 21, 24, and 28 and then weekly until day 77. On day 42 the
two cats given 200 metacercariae were given 300 more metacercariae apiece; their blood was
collected until day 9 1.
Each cat was anesthetized by ketamine, a final blood sample was taken and the cat was
killed with an intracardiac overdose of Somnotol (M.T.C. Pharmaceuticals, Hamilton, Ont.).
After midline incision, the ductus choledochus was ligated at the duodenum and the duodenum
was ligated above and below the ampulla of Vater. The gall bladder, cystic duct, common bile
duct and ductus choledochus were separated, as were the lobes of the liver. Each lobe was
sliced into 2- to 3-mm wedges and all tissues were examined for flukes. Representative tissues
were fixed in 10% formaldehyde. The spleen, pancreas and viscera were examined for worms,
lesions or enlarged nodes.
Results
The cats did not show identical or in some cases even similar clinical courses. Each
experiment will be discussed briefly, typical case histories presented and finally the
collected data will be given.
Experiment 1. Clinical signs-rectal temperature, food intake, stool quality, color,
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780 Watson and Croll
or consistency, urine output, vomiting, and behavior-were not different between
the controls and the infected cats. All infected cats passed ova from the 17th day
post-infection, and M. conjunctus was found in the biliary trees. Significant changes
(p = 0.05) in the measured parameters were examined by analysis of variance on log
transformed raw data. Differences were sought among the controls or among the
experimental cats during the infection and, finally, between the controls and experi-
mental cats on each day of sampling. Experimental cats showed: a significant
eosinophilia from days 12 to 32; a significant increase in albumin/globulin ratio; a
significant elevation in (YZ globulin, alanine aminotransferase and leucine aminopep-
tidase.
Experiment 2. Cat 6 was asymptomatic until fluke patency on day 17 when it
passed brown, watery feces without mucus, with ova. The diarrhea lasted for about
36 hours and returned irregularly until day 5 1; then it became continuous until day
59, when the cat was killed. Necropsy showed 252 adults of M. conjunctus (84%
establishment) with 80 in each lateral lobe and 24, 24, and 23 respectively in the
median lobes. No worms were found in the gall bladder or cystic duct, but 21 were
together in the common duct and extra-hepatic lobar ducts. No flukes were in the
caudal lobe.
Cat 7 followed a more spectacular clinical course and is considered a typical case
of symptomatic metorchiasis (table I). The cat was without symptoms until day 14,
when it developed scleral and dermal icterus, and the serum changed from pale straw
color to a canary yellow color. On day 14 the cat was emaciated, wet in the perianal
region, and had a rectal temperature of 38.5"C. It refused food and water, was anuric
and constipated on day 15, and had a temperature of 38.9"C. On day 16, reddish-
brown urine was expelled manually from the bladder. The cat lay in its cage, moved
only when induced, had stiff legs, and was apparently in considerable discomfort.
On day 17, 38.6 g of dense, gray feces with ova were expelled with a little yellow
mucus. The temperature was 38.9"C and a little reddish-brown urine was on the
floor of the cage; the cat ate and drank. By day 21 the cat could move slowly, ate
more vigorously, and expelled firm green feces. By day 22 the feces seemed more
normal and by day 28 the feces were normal and the serum was again straw colored.
The cat was killed on day 120.
All hepatic lobes of cat 7 were infected with M. conjunctus. There were 112 worms
in the main intrahepatic ducts of the left lateral lobe, 19 in the right lateral, 1 in the
median, 48 in the right median and 3 in the caudal lobe. One worm was in the cystic
duct, with a total of 183 adults (63% establishment).
Clinical laboratory values for cat 7 are given in table I. There was significant
eosinophilia in both cats between days 18 and 21. Leucine aminopeptidase and
alanine aminotransferase were elevated significantly in cats 6 and 7 between days 7
and 2 1 post-infection. No significant change occurred in alkaline phosphatase. Total
serum protein was stable in cat 6, but it was greatly elevated on days 18 to 21 in cat
7, which had concurrent severe clinical signs. On day 18 this elevation resulted from
increased globulin despite depressed albumin and a globulin.
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Table I. Hematological and serological findings for cat 7, inoculated with 300 metacercariae
Globulin Bilirubin
Eosinophils Prot Alb Glob Alb LAP SAP AAT
#/nun3% g/dl g/dl g/dl A/G % a p y w' ml SU/1 1U/I Tot Dir Indir
% % % mg/dl mg/dl mg/dl 5
- 2
387 2.1 6.6 3.30 3.30 1.00 50.2 29.6 11.1 18.5 5.50 0.42 39.8 0.194 -
2-
480 2.1 6.1 3.00 3.10 0.97 48.4 20.0 11.3 20.4 5.00 1.00 707.1 0.270 0.115 0.155 F'
5'
1920 7.9 7.8 3.68 4.12 0.89 47.2 23.9 12.1 16.9 8.83 2.02 348.7 7.852 5.774 2.078
5858 29.0 8.9 3.90 5.00 0.78 42.5 24.9 14.1 18.2 8.30 0.98 317.3 3.080 1.840 1.240 E i:
4746 19.1 7.7 3.50 4.20 0.83 45.6 23.5 12.7 18.0 7.16 2.30 364.6 1.210 0.650 0.560
1616 6.7 6.9 3.15 3.75 0.84 45.5 23.2 12.1 18.9 6.66 1.38 151.8 0.454 0.144 0.310
WBC
x 103
/m3
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18.5
22.3
24.4
20.2
22.6
24.1
vet.sagepub.com
post-infection; WBC = white blood cells; alb = albumin; glob = globulin; A/G = albumin: globulin ratio;
= leucine aminopeptidase; SAP = serum alkaline phosphatase; AAT = alanine amhotransferase.
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782
,OOt
I
b I
Watson and Croll
300
7 14 21 28 35 42 49 56 63 70 77 84 91
DAYS POST INOCULATION
Fig. 1: Absolute numbers of eosinophils in circulation of two cats given 200 metacercariae
of Metorchis conjunctus, per 0s on day 0 and 300 more on day 42.
Experiment 3. All infected cats started passing ova on day 17. A total of 340 adults
of M. conjunctus were in the left lateral lobes of the four infected cats, and 224 in the
right lateral lobes. Only 129 were recovered from the left median lobes and 77 from
the right; there were no flukes in the caudal lobes of any cats. The rate of
establishment varied between 40.4% and 59.7%; cat 9, which received a second
inoculum on day 24, had 202 worms (40.4% of its total inocula), and cat 10, also re-
inoculated, had 294 worms (58.5% of its total doses). Cats 11 and 12 had 179 (59.7%)
and 142 (47.3%) respectively.
Eosinophilia occurred between days 14 and 28 post-infection in all cats. Reinfection
caused renewed absolute and relative eosinophilia between days 7 and 21 after
reinfection. No clear protection from reinfection was conferred by a primary infection
six weeks earlier, although the second eosinophil peak was lower (fig. 1).
Lesions from single inoculum infections, less than 100 days old and with fewer
than 100 worms, were confined largely to the major branches of the lobar bile ducts.
The ducts were palpable from the surface of the liver and were faintly discolored,
but had no superficial nodularity or irregularity. The intrahepatic ducts were dilated
and the walls were thickened with dense fibrosis to a maximum thickness of 4 mm.
Typically, there was no extrahepatic duct involvement, but dark green or yellow
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Meforchis in Cats 783
viscous exudate with cell debris and ova occupied these ducts. The gall bladders
often were distended but the walls were not thickened. Many cats had enlarged
mesentenc lymph nodes [9], and one cat had four. The severity of gross lesions was
directly related to the intensity of the worm burden. Cat 7 had 112 worms in the left
lateral lobe, which contained bile and cell debris; extensive primary and secondary
bile duct enlargement; and conspicuous fibrosis.
Short-term patent infections of under 32 days were characterized by extensive
epithelial exfoliation. Pressure atrophy of the ducts was seen when the worms
completely filled the distended ducts. Fibrosis was restricted to the portal regions in
the borders between the bile ducts and the hepatic parenchyma. Cellular infiltrates,
rich in eosinophils but without increased lymphocytes or plasma cells, were in the
lamina propria of the dilated and hyperplastic ducts. Extrahepatic damage was
generally slight and was restricted to epithelial hyperplasia.
Cats killed between 32 and 150 days post-infection also had extensive exfoliation
of the biliary duct epithelial cells. There was widespread hyperplasia of the biliary
epithelium and large areas of adenomatous tissue. Intense eosinophilic infiltrates
were replaced by mononuclear cells in the more chronic infections.
Cats 8 and 9, reinfected 42 days after primary infection, showed both chronic and
acute lesions when killed on day I13 post-infection. Chronic lesions generally were
characterized by extensive proliferation of connective tissue, which replaced adeno-
matous hyperplasia. Another cat, given 300 metacercariae and killed on day 719
post-infection, had granuloma formation around the ova. Ova often were surrounded
by inflammatory cells, obstructing the intrahepatic bile ducts.
Discussion
Almost all cats with M. conjunctus had only brief diarrhea and anorexia. Classical
features of acute hepatic distress such as icterus, listlessness, abdominal tenderness,
steatorrhea, fluid and fecal retention, emaciation and severe diarrhea were found
only in cat 7. These results are consistent with previous observations on cats and
dogs [l, 2,6, 8, 11, 13, 23, 251. Worm burdens could not be directly related with the
extent of eosinophilia, but numbers of leukocytes fluctuated widely in both infected
and uninfected cats. The excitable nature of the cat has been related to the substantial
elevations; even 40,000 cells/ml may be normal in a cat under stress [ 191.
Peak eosinophilia was associated with maturation and oviposition of the flukes. In
other studies, C. sinensis in rabbits became patent by day 22 [25], and eosinophilia
coincided with maturation of C. sinensis in guinea pigs [20]. Eosinotactic substances
may be secreted by Opisthorchis viverrini [3], and mobilization of eosinophils followed
the migration of F. hepatica into the biliary system [15,22].
Hyperproteinemia, with elevated albumin and globulin, was seen in cat 7 (table I).
Elevated albumin could follow dehydration and shock in the acute infection. Globulin
changes reflect products of the reticulo-endothelial system; elevated ,8 globulin
is also indicative of obstructive jaundice. Both a and ,8 globulins were elevated in
rabbits infected with C. sinensis [25]. Significant changes in al, a2 and total a globulin
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784 Watson and Croll
occurred in Experiment 1. These changes generally are associated with inflammatory
reactions and occur in obstructive jaundice, probably because of increased serum
lipid [6, 181. Changes in y globulin were inconsistent in both infected and uninfected
cats. This may explain the apparent lack of resistance to re-infection seen in
Experiment 3. There was a good correlation between the albumin and globulin
change and infection (R = 0.7884; PR>R = 0.001). The a1bumin:globulin ratio was
depressed in cat 7 and there was a tendency for it to be depressed, though not
significantly, in infected cats in Experiment 1.
Leucine aminopeptidase is concentrated in the biliary epithelium and is even more
specific as an indicator of biliary disease than alkaline phosphatase. It increased
significantly in infected cats. Alanine aminotransferase (SGPT) increased signifi-
cantly between days 6 and 16 post-infection, suggesting acute hepatocellular damage,
even in the absence of clinical signs. While this is an excellent indicator of hepato-
cellular lesions, it also may be elevated in muscle or cardiac damage. Serum bilirubin
was elevated significantly only in cat 7, which had severe icterus. The lack of
conclusive hematological or serological abnormalities in metorchiasis in man [7]
probably results from the transitoriness of many of these changes. In man, the acute
clinical features of opisthorchiasis and clonorchiasis are brief [lo, 14, 21, 261.
Direct mechanical effects can be seen in histological sections. The attachment of
the suckers to the biliary epithelium, adherence of hepatocytes and inflammatory
cells to the worms, atrophy of the epithelium and the presence of host cells in the
parasite's caeca all indicate direct mechanical damage. Lesions ascribed to natural
infections of M. albidus in cats in Europe are similar to those seen in our experiments
116, 231.
Acknowledgements
The authors wish to thank the Natural Sciences and Engineering Research Council of
Canada for a grant to Neil A. Croll, which partially financed this project. Research at the
Institute of Parasitology is supported by N.S.E.R.C. and the Formation des Chercheurs et
d'Action ConcertCe du Ministere de l'Education du Quebec.
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