A model of hepatic amoebiasis in random bred mice

346 TRANSACTIONS OF THE ROYALSOCIETY OF TROPICAL MEDICINEAND HYCENH(1989)83, 346-348

A model of hepatic amoebiasis

in random

bred mice

K. C. Bhol, R. M. Mukherjee, S. Mehra, T. K. Maitra and K. N. Jab’ Gastroenterology, CMRI, 712 Diamond Harbour Road, Calcutta 700027, India Abstract This study describes a model of hepatic amoebiasis in random bred mice (MF2). Mice were infected by introducing liver tissue from hamsters (Mesocricetus auratus), containing about 5~ lo4 trophozoites of Entamoeba histolytica, between adjacent liver lobes. Direct inoculation of xenic E. histolytica resulted in mortality within 24 h, whereas monoxenic and axenic strains failed to produce any lesions. Serial mouse liver passage resulted in increased lesion score, numb& of metastatic abscesses,and mortality. Metronidazole 150 ma/kg nroduced comnlete healinn of the abscess. It is expected that this model wx be useful to study host-parasite interactions, immunology and experimental chemotherapy of amoebiasis. Introduction Various models of hepatic and intestinal amoebiasis in different animals have been described (MEEROVITCH & CMDEE, 1988). However, it is difficult to infect mice with Entamoeba histolytica; experimental amoebiasishas been described only in mice which are genetically susceptible or immunocompromised (GHADIRIAN 81 KONGSHAVN, 1984; WIJESUNDERA, 1980; STERN et al., 1984). WESTPHAL & MICHEL

(1970) were able to produce amoebic abscessesin the mesentery of mice by intraperitoneal inoculation of axenic E. histolytica, but their successwith intrahepatic inoculation was limited. This study describes a model of hepatic amoebiasis in random bred mice (strain MF2) produced by inoculating small pieces of liver abscesstissue from hamsters between the lobes of the liver in a manner similar to that described by DAS et al. (1985). It is expected that the experimental model described will be useful for studying host-parasite interaction, immunology and experimental chemotherapy of amoebiasis. Materials and Methods Experimental animals Four to 5 weeks old golden hamsters (Mesocricetus aureus) weighing 40-50 g, and 5-7 weeks old mice (strain MF2), weighing 20-25 g, of either sex, from our own breeding colony were used for experiments. The animals were maintained on a normal laboratory diet and water ad libitum. Infecting inoculum A pathogenic strain of E. histolytica of human origin (strain KCG:0986: 1l), maintained xenically in TYSGM-9 medium (DIAMOND, 1982) and monoxenically or axenicallv in BIS-33 medium (DIAMOND et al.. 1978), was *used. The associate & the monoxenii media was Crithidia (obtained from Dr H. J. Bos ‘Author for correspondence.

Korhari

Cenrre of

through Organon Research Centre, Calcutta). This strain was desianated strain REF IPRR bv Dr L. S. Diamond (Desartment of Health and I&man Services, NIH, Bethesda, USA). Suspensions were prepared in phosphate-buffered saline (PBS, pH 7.2) from 72 h cultures of E. histoZytica.The inoculum size was adjusted to about 50 000 trophozoitesl 0.05 ml, and hamsters were inoculated as described by DUTTA (1970). On the 6th day after infection, the hamsters were killed and liver abscesstissue removed for inoculation into mice. Infection and examination of mice An inocuhun of O-05 ml containing about 50 000 trophozoites of xenic, monoxenic or axenic E. histolytiza was injected directly into the right lobe of the liver of mice through a 26 gauge needle. Small pieces of infected hamster liver tissue containing approximately the same number of active trophozoites were placed between adjacent liver lobes of different group of mice after laparotomy under light ether anaesthesia. The inocuhun size was determined by homogenizing the piece of liver abscess tissue in PBS by repeated suction and aspiration with a Pasteur pipette and then counting the -uophozoites with a haetiocytometer. In control mice. O-05 ml of bacterial or Crithidia suspensions from cultures, pieces of normal liver or uninfected pieces of liver from infected hamsters or mice, were inoculated in a similar manner. In all these experiments, scrupulous asepsis was maintained. F&r animais of e&h group were killed on the 7th, 14th. 21st. 28th and 35th davs after infection and thek he; abscesseswere scored by the method described earlier (DUTTA, 1970). The virulence index for hepatic lesions was determined by modifying the formula of VINAYAK et al. (19811 as follows: (Total scoresof animals &ith hver lesions + maximum possible score)x 100 Liver lesions, when present, were smearedin PBS for microscopic examination and inoculated to culture medium TYS-GM-9 for recovery of trophozoites. Also. infected liver tissue was tied in buffered form01 salin’e for histological examination. Metronidazole treatment In a separateset of exoeriments, mice were infected similarly-but were given a suspension of metronidazole (Sinma in 0.2% Bactoaaar (Difco) orallv in differ&tdoHes (50, 100, 150, &d 200 mi/kg daily) for 4 d, starting on the day of infection (day 0). Control and experimental animals were killed on the 7th day after infection and hepatic lesions were scored. Antibody response On each day when mice were killed, blood from infected and control animals was collected from the

347 1). None of the animals of the different control groups showed any hepatic lesion. Motile E. hiswlyticu trophozoites were seen in smearsof the liver abscessesand were recovered from cultures inoculated with abscesstissue. Histological sections showed the characteristic lesions of amoebic liver abscess.

retro-orbital plexus by narrow-tipped Pasteur pipettes. Serum was separated and anti-E. hisw&icu antibody was detected by indirect haemagglutination assay (IHA) (KAGAN & NORMAN, 1970, 1974). Results Effect of intrahepatic inoculution w mice of xenic, monexenic and axenic cultures of E. histolytica

Effect of serial passage in mouseliver

All animals died on the 2nd day after inoculation of

With successivepassagesthrough mouse liver the lesion scoreincreased, with development of metastatic abscessesand an increase in mortality (Table 2), suggesting enhancement of virulence of the E.

E. ht&!yrica grown in xenic culture or the associated

bacteria. The monoxenic and the axenic strains failed to produce any abscessin the liver and none of these animals died.

hiswlytica. E&i

of hamster liver abscess tissue into

;~~~culation

Effect of metronidazole on hepatic abscessproduction in mice

Most of the infected mice developed hepatic abscesseswith an average lesion score of 3.45 (Table Table

1. Results of inoculating

Days after infection

A dose of 150 mg/kg of metronidazole prevented

mice with hamster liver tissue containing

No. infected/no. inoculated

No. dead/no. killed

1:

414

o/4 113

2 35

414 314 314

212 014 212

Table 2. Effects of mouse liver passage on virulence

No. of passage

Days after infection

Liver lesion score (meant-SE)

of Entamoeba

No. dead/no. killed

l/l 414

Liver lesion score (mean?SE)

Virulence index(%)

014

100 100 100 82.1 91.6 95.4 100

6/7

818

612 512

4 +o 3.5 kO.28 4 +o 3.28kO.54

4-5 6-7

616 11111

313 714 3/o

3.6 +0*21 3.8 f0.12 4 +o

l/10

313

El

4! 128 512 1024

hktolytka

6-7 8-9 IO-11

ML-2b

Aver;gAre$pl

3.5 kO.28 3.75kO.25 4 *o 3 +1 3 +1

No. of animals infected/no. of animals inoculated

4-5

ML-la

Enfamoeba bisfofytica

None survived

*First passagethrough mouse liver. bSecond passagethrough mouse liver. Table

3. Effect of metronidazole

Group

Dose of metronidazole”

on hepatic amoebiasis

No. cured/no. inoculated

in mice

No. showing liver lesions of grade 0 1 2 3 4 2

3.8

Activity (%j

:

Nilb 50

0110 s/10

:

100 150

loll0 8/10

108

-2

-

-

-

0 ;:;

100 ii00

200

lO/lO

10

-

-

-

-

0

100

5 ‘mg/kg. bControl group.

;I;28

Average score

0

348

abscessformation in all animals. Doses less than this resulted in only a reduction in abscess incidence (Table 3). Antibody response

The antibody titre of infected mice was 1:8 in the first week; it increased gradually and reached a maximum of 1:1024 in the 5th week after infection, when the animals had an average lesion score of 4. Discussion It has generally been thought that mice are naturally resistant to E. kistolyticu infection. In all previous studies, genetic composition and the immunological state of mice have been important factors in the induction of caecal or hepatic amoebiasis. Although WESTPHAL 8~ MICHEL (1970) could produce mesenteric abscessesafter intraperitoneal inoculation of axenic E. kistolyticcf, their success with intrahepatic inoculation was lirmted. Out of 9 inbred and 1 outbred strains of mice, GHADIRIAN81 KONGSHAVN(1984) could produce caecalamoebiasisin only 6 inbred strains (C3H/HeCr, BALB/c, NZB BIN, BIOA, DBA/Z, C57BL/6), and no lesion could be produced in the only random-bred mouse (CD-l) used in their study. To the best of our knowledge, this is the only study in which a consistent, reproducible model of hepatic amoebiasis has been produced in a random bred mouse. To achieve this it was necessary to induce hepatic abscess in the hamster first and then to introduce the infected hamster liver tissue in between the liver lobes of mice. Direct inoculation of xenic cultures produced mortality within 24 h, probably due to bacterial infection, and monoxenic and axenic E. kisrolytica did not cause abscessformation. This suggeststhat random bred mice are relatively resistant to E. kistolytica infection and abscessformation is dependent upon the virulence of the organism. Prior passage of organisms in hamster liver could have enhanced the virulence, since an increase in virulence of E. kiszot’yticu after liver passageis well documented (LUSHBAUGHet al.. 1978). Our own studv further confirms such an enhancement of virulence. After several liver passagesin mice we also observe an increase in lesion score, metastatic abscessesand mortality. The findings further suggestthat, although the defence mechanism of this strain of mouse is able to contain infection by moderately virulent E. kistolyricu, there is a breakdown of this defence barrier by a virulent variant of the same strain. The curative activity of metronidaxole in a doserelated manner also confirms the validity of this model. The use of the mouse has certain advantages over the existing hamster model for experimental chemotherapeutic studies. The amount of drug required is lessand maintenance of random bred mice is easy. Since the immune processes of mice can be related to those in humans, it is expected that experimental amoebiasisin this random bred mouse will lead to a better understanding of the immunological mechanisms involved in human amoebiasis.

Acknowledgements

We are grateful to Dr L. S. Diamond, Project Collaborator, Department of Health and Human Services, National Institute of Health, Bethesda, Maryland 20205, USA (shared cost project No. 01-160-N), for his helpful criticism and advice. We thank Dr D. P. Halder, Department of Zoology, Kalyani University for suggestionsand encouragement, and Mr E. G. P. Nair for secretarial assistance. References

Das, P., Narain, L., Dutta, G. P. & Pal, S. C. (1985). improved method of producing amoebic.liver abscessin hamster for screening of systemically active amoebicides. Australian Journal of Experimental Biology and Medical

Sciences,63, 85-89. Diamond, L. S. (1982). A new liquid medium for the xenic cultivation of Entamoeba hiswlytica and other lumen dwelling protozoa. Journal of Parasitology, 68, 958-959. Diamond, L. S., Harlow, D. R. & Cunnick, C. (1978). A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Transactions of the Royal Society of Tropical Medicine and Hygiene, 72, 431-432.

Dutta, G. P. (1970). Amoebic liver abscessproduction in hamster bv intraneritoneal inoculation of troohozoites of Entamoeba histot$ica without laparotomy. &ceedings of the Indian National ScienceAcademy, MB, 99-101. Ghadirian, E. & Kongshavn, P. A. L. (1984). Genetic control of susceptibility of mice to infection with Entamoeba hiswlvtica. Parasite Zmmunoloev. 6, 349-360.

Kagan, I. G. & N&man, B. L. (1970). S&diagnosis of parasitic disease. In: Manual of Clinical Microbiology, Blair, J. E., Lennette, E. & Truant, T. P. (editors). American Society for Microbiology, pp. 455-486. Kagan, I. G. & Norman, L. (1974). Serodiagnosis of parasitic disease. In: Manual of Clinical Microbiology, Lennette, E. G., Spaalding~ N. H. & Truant, T. P. (editors), 2nd edition. Amencan Society for Microbiology, pp. 654663. Lushbaugh, W. B., Kairalla, A. B., Loadholt, C. B. & Pittman, F. E. (1978). Effect of hamster liver passageon the virulence of axenicallv cultivated Entamoeba histolvtica. American journal

of .Tropical Medicine and Hygikte,

27, 248-254. Meerovitch, E. & Chadee, K. (1988). In vivo models for pathogenicity in amoebiasis. In: Amebiasis, Human Infection by Entamoeba histolytica, Ravdin, J. I. (editor), 1st edition. New York, N.Y.: John Wiley, pp. 177-190. Stem, J. J., Graybill, J. R. & Drutz, D. J. (1984). Murine amoebiasis: the role of the macrophage in host defence. f7;T8gt

Journal of Tropical Medicine and Hygiene, 33,

Vinayak, V.’ K., Sanhney, S., Jain, P., Chugh, S., Naik, S. R. & Chakravarti, R. N. (1981). Virulence of Entamoeba histolytica in rat and its comparison with the serological response of the amoebic patients. Transactions of the :2y;;

Society of Tropical Medicine and Hygtene, 75,

Westpha!; A. & Michel, R. (1970). Versuche zur extraintestinal an Entamoeba hiswlytka infektionen der maus and anderer Nager. Zeitschrift fiir Tropenmedizin und Parasiwlogie,

21, 383.

Wijesundera, M. de (1980). Hepatic amoebiasis in immunodepressedmice. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 216-220. Received 1 June 1988; revised 16 August 1988; accepted

for publication 30 September 1988