Entamoeba histolytica in inbred mice: evidence of liver involvement

617

TRANSACTIONSOF THE ROYAI. SOCIETYOF TROPICALMEDICINE AND HYGIENE (1987) 81, 617-620

Entamoeba

histolytica in inbred mice: liver involvement

evidence

of

D. G. OWEN MRC Experimental Embryology and Teratology Unit, WoodmansterneRoad, Carshalton, Surrey, SMS 4EF, UK Abstract Following observations that certain mouse strains appeared to be susceptible to liver invasion by Entamoebahistolytica, mouse strains CBAEIN, CBA/HN, nu/nu, CBA/HN nu/+, CBA/HN Dh/+, CBA/HN +/+, C3H/HeJ Lps/Lps and ABIH, were infected intra-intestinally with strain SAW 408 (zymodeme II) of the amoeba. A number of mice from each group died during the fist 14 days with acute lesions. The remainder were killed at 4 and 6 months after infection. CBAIHN, CBA/HN Dh/+ . and CBA/HN +I+ mice all had amoeba-infected livers, although only 2 mice had extensive, typicai liver lesions. Introduction There have been many attempts to develop a suitable rodent model host for the study of hepatic amoebiasis but spontaneous liver invasion resulting from caecal infection has not been satisfactorily achieved. Abscesseshave been produced in neonatal hamsters (MA-I-TERN & KEISTER, 1977; GHADIRIAN & MEEROVITCH, 1979; CAPIN et al., 1980) by the inoculation of axenic amoebaedirectly into the liver. Liver invasion has been achieved spontaneously in mice only when subjected to severe immunosuppression (WIJESUNDERA, 1980). GOLD & KAGAN, (1978) failed to show strain susceptibility in 9 mouse strains; only 3.8% of 209 mice inoculated directly into the liver with axenic strain HK9 had lesions or live amoebae 3-7 days later. Amoebae have been found in the livers of CBA/I-IN Dh/+ and NZB mice (OWEN & ACKERS, 1982; OWEN, 1985) following intracaecal inoculation with the highly invasive polyxenic strain SAW 408 (zymodeme II, SARGENJNT &WILLIAMS, 1978). Few of these susceptible mice survived infection, possibly because of their sensitivity to the bacterial flora associatedwith the amoebaein culture. Bacteria have long been known to have a potentiating effect on amoebae (PHILLIPS et al., 1955; WITNER 8z ROSENBAUM, 1970; PHILLIPS et al., 1972; Bos & HAGE, 1975). In a previous study, susceptible mice given an amoeba-freebacterial suspension from polyxenic cultures failed to show clinical symptoms, even if athymic. Groups of 12 ONU mice and 3 to 5 C3Wmg CBA/HN and CBA/HN Dh/+ mice all survived intraintestinal inoculation with bacterial suspensions from cultures of 2 E. histolytica strains, SAW 408 and SAW 760 (zymodeme IX) (unpublished observation). The work described here takes advantage of the (so far unexplained) fact that amoebaeisolated from the caecum of rats, dosed orally with SAW 408 6 months previously, could be inoculated intracaecally to susceptible strains of mice with greatly reduced mortality (OWEN, 1984). This change in pathogenicity could have been due to a modification of the bacterial flora; Presentaddress:London School of Hygiene and Tropical Medicine, WinchesFarm Laboratories,395Hatfield Road, St Albans, Hertfordshire,

AL4 OXQ, UK.

the invasiveness of the organisms appeared to be relatively unaffected. Groups of CBA/I-IN strain mice carrying asplenic and athymic genes were dosed with the apparently modified amoeba culture and kept for up to 6 months to acertain whether consistent, spontaneous liver invasion occurred, and if so whether it would develop into clinical lesions. Materials and Methods Mice Mice were obtained from the MRC Experimental Embryology and Teratology Unit microbiologically defined (MD) breeding colony and consisted of 12 CBA/HN Xid!Xid (B cell-deficient), 7 CBA/HN-nu/nu (athymic), 10 CBAkINnu/+, 6 CBA/HN-Dh/+ (asplenic), 6 CBA/I-IN-+/+, 17 C3I-VHeJ lps/lps (macrophage deficient), and 9 AB/H (Biozxi high responder). All animals were maintained in a negative pressure plastic iilm isolator with filtered air and all materials, including diet, water, and bedding, were triple wrapped, sealed, irradiated at 2.5 Mrad and introduced into thFdisolator via an entry port sterilized with 2.0% peracetic Amoebae The culture strain SAW 408, zymodeme II (SARGEAUNT81 WILLIAMS, 1978, 1979) was kindly supplied by Mr P. Sargeaunt of the London School of Hygiene and Tropical Medicine. It was maintained in 6 ml screw capped glass culture bottles containing Robinson’s diphasic medium (ROBINSON, 1%8) and passaged 3 times weekly. 2 cultures of this strain were used, the original SAW 408 and the culture isolated from rats 6 months after infection, termed SAW 408(M). Inocularion of E. histolytica suspensions 3-d-old cultures were agitated gently until amoebae and starch granules were in suspension, pooled, centrifuged at 2000 rpm (700 g) for 5 min and the majority of the supematant discarded. The volume was adjusted to give approximately 2 X lo5 organisms per ml. Mice were anaesthetixed using halothane (Fluothane and Fluotech, I.C.I., Macclesfield, Cheshire, UK). A small incision through the skin and the abdominal wall allowed 0.1 ml amoebic suspension to be inoculated into the distal portion of the ileum. The body wall was closed with Mersilk and clamped with Michel clips. Gentamycin 4mg/O.lml was injected subcutaneously close to the suture site. Procedure Groups of all 5 mouse strains were given SAW 408(M) and groups of 6 CBAIHN and 4 AB/H were given the original

618

E. histotjka IN MICE: LIVER INVOLVEMENT

Tabk-Caecal infection with Entamoebahistolytica SAW 408 and SAW 408 (M) in some inbred mouse strains over a period of 6 months

Mice ;~~rg~~s408 Strain CBA/HN CBA/HN CBA/HN CBA/HN “cm;

No. of mice 12 nu nu nu/+ Dh/+ +/+

dying during first 14 days

1: t 17 9

ABH CBAHN AB/H *Positive liver cultures

:

: 4 Control mice receiving SAW 408

x

SAW408(Table 1). All micefound deador moribund were examinedpost-mortemand liver sampleswere taken into Robinson’smedium.Half the survivorsin eachgroup were killed 4 months, and the remainder 6 months, after infection. At post-mortemexamination samplesof liver were cultured in Robinson’smedium, and someliver and caecal tissue was 6xed in 10% form01 saline, dehydrated, wax embedded and sectioned at 2 pm. Sectionswere stained with haematoxylin and eosin or Giemsa’s stain. Results

Both groups receiving SAW 408 died within the lirst 7 days, with acute amoebic ulceration of the caecum. Mortality was also high among the groups receiving SAW 408 (M), but small groups of mice of 5 straios were still healthy when killed 4 months after infection, aswere the mice of 4 strains killed 6 months after infection (Table). All animals dying during the first 14 days were acutely infected. All those killed at 4 and 6 months had typical chronic lesions, varying only slightly in degree between individuals. The caecum was shrunken, oedematousand pale, almost translucent and lilled with clear mucus containing large numbers of active trophozoites. None of the animals displayed any clinical symptoms and all had normal stools. Trophozoites were isolated from apparently unaffected mouse liver samples by passageinto Robinson’s medium from both CBA&IN Dh/+ mice sampled 4 months after infection. At 6 months, CBAHN and CBAIHN +I+ strains had positive liver cultures and one CBA&IN and one CBAEIN +/+ had extensive liver abscesses.There were no adhesions between the chronically affected caecumand the liver, and sections of the affected liver revealed numerous trophozoites around the periphery of an extensive abscess(Figs 1 & 2). In both individuals the liver contained numerous foci, enerally of interconnecting roughly circular areasof necrosis surrounded by large areas of fibrotic tissue, and in the CBAHN +I+ mouse large numbers of active trophozoites appearedto be invading the rapidly diminishing areas of normal liver tissue. Discussion

:.

(M) Number animals positive at 4 months 2/4* 315 212 2/2*

These experiments confirm earlier experiments (OWEN, 1985) in which another strain of E. histo&i-

x

Number animals positive at 6 months 3/5* 2%

4/5 415

G* 415 -

-

-

ca, SAW 760 (zymodeme IX), from the same source was used. Although not invasive for man according to the classification of SARGEAUNT 81 WILLIAMS (1978), SAW 760 induced acute and chronic lesions in susceptible mouse strains, though with a much lower mortality than SAW 408. Early experiments were all short-term, concentrating on development of the diseasein the caecum, but on 2 occasionsin CBAiHN and NZB mice organisms were observed migrating directly from acutely affected caecum into liver at a point of adhesion. Pooled liver samples from all mouse strains were routinely cultured in Robinson’s medium but only CBAHN and NZB produced positive cultures from apparently unaffected liver. In the present experiments no adhesion was seenin any of the mice with either inapparent infection or hepatic abscess.Invasion of the liver by direct contact with the infected caecum could be regarded as a chanceoccurrence, but the production of liver abscess without any accompanying adhesion indicates transport of the organisms in the blood stream. The host usually appears to have an efficient system of resistance to liver invasion and in the mouse strains tested in this laboratory only those with B-cell abnormality have consistently shown liver involvement. NZB mice have a B cell hyperplasia and are a model for immune haemolytic anaemia (BORASCHI & MELTZER, 1979). Initially the CBA/HN Dh/+ asplenic gene was thought to be the sole cause of susceptibility to liver invasion, but CBA&IN mice have a naturally occurring B cell deficiency (SCHER, 1981; SCHER et al., 1976) which could be important in susceptibility to liver invasion. Both the asplenic and athymic (r&m) geneshave been backcrossedmore than 6 generations on to the CBAHN strain, and the apparent sensitivity of these mice may be due to the background strain. The athymic nude mouse, although susceptible to caecal infection, has not been the most consistent in producing chronic lesions, and in previous studies failed to sustain large intrahepatic inoculations of the axenic amoeba HK9, confirming the findings of STERN et al. (1984) who used another axenic amoeba (HM-I-IMSS) in BALB/c nu/nu mice. In this laboratory, in experiments using athymic mice with the CBAEIN background the homozygotes were consistently susceptible to both caecal and liver invasion.

D. G. OWEN

Fig. I. Affected liver showing typical abscess.

Fig. 2. E. hi.dytico

trophozoites at the edge of a lesion.

619

E. histolytica IN MICE: LIVER INVOLVEMENT

620

The remaining groups of mice appeared not to be susceptible to liver infection. The important role played by macrophages as a defence against amoebiasis has been shown bv STERNet al. (1984). C3H/He J mice had been considered to be a potentially useful model host, but invasion of liver was not observed in this strain. Although susceptible to caecal invasion, AB/H mice also failed to produce any discernible liver infection. GOLD & KAGAN (1978) failed to find mice of 9 strains susceptible to liver inoculation, using 4 different axenic amoebae strains activated before inoculation with Bacteroides mbiosus. However, these workers found pronounced multiplication of oreanisms in the liver of CBA mice, but these were noi successfully passaged further.. Recently, WIJESUNDERA (1980) found that immunosuppression with cyclophosphamide and with anti-mouse lymphocyte sera resulted in the development of consistent, chronic lesions in the caecum of mice of the very resistant mouse strain (C3H/mg, 6-9 days after infection, and also resulted in early hepatic lesions in 5 mice in the 9-day group. CAPIN et al. (1980) found that complement depletion, also, was an important factor in improving the frequency and severity of hepatic lesions in hamsters. The mechanisms involved in susceptibility of some individuals to liver invasion by E. histolytica are largely unknown. It appears that CBA/HN mice are a potentially useful strain for the study of the natural progression of the disease from caecum to liver. They have a very specific immunodeficiency, a particular late-developing B lymphocyte population responsible for the passage of signals between macrophages and T cells. A closer study may well define some of the factors responsible for resistance to liver invasion. References

Bos, H. J. and Hage, A. J. (1975). Virulence of bacteria associated, crithidia associated and axenic Entamoeba his~oIvtica: exoerimental hamster liver infections with stra& f&m patients and carriers. Zeitschrifi fir Parasitenkunde, 47, 79-89. Capin, R., Capin, N. R., Carmona, M. & Ortiz, L. (1980). Effect of complement depletion on induction of amoebic liver abscess in the hamster. Arckiws de Znvestigucion Medica (Mexico), 11, (Supplement l), 173-180.

Ghadirian, E. & Meerovitch, E. (1979). Pathogenicity of axenically cultivated Entamoeba kistolytica strain 2OO:NIH in the hamster. 7ounzal of ParaFitoloav, -. 65,

768-771. Gold, D. & Kagan, I. G. (1978). Susceptibility of various strains of mice to Entamoeba hiswlytica. Journal of Parasitology, 64, 937-938.

Mattern, C. F. T. & Keister, D. B. (1977). Experimental arnoebiasis. II. Hepatic amoebiasis in the newborn hamster. American Journal of Tropical Medicine and Hygiene, 26, 402-411. Owen? D. G. (1984). Attempts at oral infection of rats and race with trophozoites of Entamoeba kistolytica. Transactions of the Royal Society of Tropical Medicine and Hygiene, 78. 160-164. Owen; D. G. (1985). A mouse model for Entamoeba histolytica infections. Laboratory Animals 19, 297-304.

Owen, D. G. & Ackers, J. (1982). The susceptibility of various inbred mouse strains to intra-intestinal infection with Entamoeba kistolytica. Parasiwloty, 85, (abstract). Phillips, B. P., Wolfe, P. A., Rees, C. W., Gordon, H. A., Wright, W. H. & Reyniers, J. A. (1955). Studies on the amoeba-bacteriarelation&p in amoebiasis. Comparative results of the intracaecal inoculation of germfree, monocontaminated and conventional guinea pigs with Entanweba kiswlytica. AmericanJournul of Tropical Medicine and Hygiene, 78, 116-126. Phillips, B. P., Diamond, L. S., Bartgis, I. L. & Stuppler, S. A. (1972). Results of intracaecal inoculation of germfree and conventional guinea pigs and germfree rats with axe&ally cultivated Entamoeba kistolytica. 3ournal of Protoeoology, 19, 498-499. Robinson, G. L. (1%8). The laboratory diagnosis of human parasitic amoebae. Transactions of the Royal Society of Tropical Medicine and Hygiene, 62, 285-294. Sargeaunt, P. G. & Williams, J. E. (1978). The differentiation of invasive and non-invasive Entamoeba hiswlytica by isoenzyme electrophoresis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 72, 519-521. Sargeaunt, P. G. & Williams, J. E. (1979). Electrophoretic isoenzyme patterns of the pathogenic and nonpathogenic intestinal amoebae of man. Transactions of the Royal Sociery of Tropical Medicine and Hygiene, 73, 225-227. Scher, I. (1981). B-lymphocyte development and heterogeneity: analysis with the immune-defective CBA/N mouse strain. In: Zmmunological defects in laborawty animals, Gershwin, M. E. &Merchant, B. (editors). New York and London: Plenum Press, pp. 163-185. Scher, I., Shannon, S. 0. & Paul, W. E. (1976). X-linked B-lymphocyte defect in CBA/N mice. III. Abnormal development of B-lymphocyte populations delined by their density of surface immunoglobin. 3oumal of Exper&ntal Medicine, 144, 507-518. Stern, J. J., Greybill, J. R. & Drutz, D. J. (1984). Murine amoebiasis: the role of the macro@age in host defense. t7zr Journal of Tropical Medzme and Hygiene, 33, Wijesundera, M. de S. (1980). Hepatic arnoebiasis in immunodepressed mice. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 216-220. Wittner, M. & Rosenbaum, R. M. (1970). Role of bacteria in modifying virulence of Entamoeba kiswlytica. Studies of amoebae from axenic cultures. American 3ountul of Tropical Medicine and Hygiene, 19, 755-761. Accepted for publication 7 April 1986