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Repeated Oral Dosing withListeria monocytogenesin Mice as a Model of Central Nervous System Listeriosis in Man
J. Comp. Path. 1999 Vol. 121, 117–125
Repeated Oral Dosing with Listeria monocytogenes in Mice as a Model of Central Nervous System Listeriosis in Man J. Altimira, N. Prats, S. Lo´pez, M. Domingo, V. Briones∗, L. Domı´nguez∗ and A. Marco Departamento de Patologı´a y Produccio´n Animales (Histologı´a y Anatomı´a Patolo´gica), Facultad de Veterinaria, Universidad Auto´noma de Barcelona, 08193 Bellaterra (Barcelona) and ∗Departamento de Patologı´a Animal I, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain Summary Human listeriosis is a food-borne disease of immunosuppressed or previously healthy adults. The repeated oral administration of a sublethal dose (5×109 colony-forming units) of Listeria monocytogenes for 7 or 10 consecutive days led to the development of severe central nervous system (CNS) lesions in 25% of experimental mice. Histopathological examination of the brain revealed rhombencephalitis and ventriculitis as two distinct inflammatory patterns, resembling those seen in human listeriosis. This model would seem to be potentially useful for research on pathogenesis, predisposing factors and therapy in CNS listeriosis in man. 1999 W.B. Saunders Company Limited
Introduction Infection of the central nervous system (CNS) by Listeria monocytogenes is currently considered one of the most common forms of human listeriosis particularly in the newborn and in immunosuppressed patients; it sometimes occurs, however, in previously healthy persons (Ho et al., 1986; McLauchlin, 1990; Farber and Peterkin, 1991). Human listeriosis is regarded as a foodborne disease (Farber and Peterkin, 1991; Schuchat et al., 1991; Lorber, 1997) and food products with an overgrowth of L. monocytogenes have sometimes been incriminated as the source of infection (Schlech et al., 1983; Farber and Peterkin, 1991). However, there is still debate as to why only a small proportion of healthy persons exposed to a contaminated food source develop the disease (Farber and Peterkin, 1991). Three different patterns of inflammatory lesion in the CNS have been described in human listeriosis. Meningitis is the most common form in immunosuppressed and neonatal subjects but is also occasionally reported in previously healthy persons (McLauchlin, 1990; Schuchat et al., 1991; Skogberg et al., 1992). Rhombencephalitis has been reported in adults (Bach and Davis, 1987; Armstrong and Fung, 1993; Kirk, 1993). Choriomeningitis and periventriculitis have also been described, sometimes in Correspondence to: A. Marco. 0021–9975/99/060117+09 $12.00
1999 W.B. Saunders Company Limited
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association with septicaemic lesions in organs outside the CNS (Yamauchi et al., 1978). Listerial rhombencephalitis has often been described in adult sheep and cattle and there is evidence that L. monocytogenes passes along cranial nerves and causes encephalitis, mainly affecting the brain stem (Charlton and Garcı´a, 1977). The aim of the present study was to develop an experimental model in which CNS lesions mimicking those seen in the human species were reproduced. In previously described murine models, highly artificial methods of infection (intracranial, middle ear, subcutaneous or intravenous) were used (Leist et al., 1988; Prats et al., 1992; Berche, 1995; Blanot et al., 1997). In our opinion, these methods, with their devastating effects, do not represent an accurate experimental model for natural human listeriosis, in which oral infection is commonly assumed. Outbreaks of human listeriosis sometimes last for weeks or months, suggesting prolonged exposure to the bacteria (Schuchat et al., 1991). It would seem, therefore, that patients may consume a contaminated food (e.g., raw vegetables, soft cheese) for some time before showing clinical signs. To mimic this situation, mice were given daily oral doses for varying periods. In this way it was possible to study CNS lesions produced by multiple sublethal doses of the organism. Materials and Methods Mice Swiss CD1 mice (Panlab, Barcelona) of either sex, aged 6 weeks and weighing 20 g, were used. Four groups (O, A, B and C) were established. Mice of group O (n=6), given a single oral dose of L. monocytogenes, were humanely killed for necropsy in subgroups of three on days 4 and 7 after infection. Mice of groups A, B and C (n=12/group) were dosed orally once each day for 4, 7 and 10 consecutive days, respectively, and killed in subgroups of four for necropsy on days 1, 4 and 7 after the final dose. (The third subgroup of group B consisted of only three mice, because one animal had died.) Bacterial Inocula Strain P-14B of L. monocytogenes serovar 4b, of human origin, was grown on brain–heart infusion agar (BHI; Difco, USA) at 37°C for 22 h. The organisms were centrifuged and resuspended in sterile 0·9% saline. The concentration of the cell suspension was adjusted spectrophotometrically at 550 nm and diluted to obtain the desired daily inoculum of 5×109 colony-forming units of L. monocytogenes; this was administered orally by a feeding bottle as previously described (Marco et al., 1997). Histopathological and Immunohistochemical Examination of Samples Samples of liver, spleen, lymph nodes (mesenteric, maxillary and lumbar), stomach, small and large intestine (several samples from each), kidney, adrenal gland, urinary bladder, reproductive organs, lung, heart, thymus and brain were obtained immediately after euthanasia, fixed for at least 48 h in 10% buffered formalin and embedded in paraffin wax by routine procedures. Serial sections (4 lm) were
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stained with haematoxylin and eosin (HE) or processed for immunohistochemical examination by the avidin–biotin complex (ABC) method, as previously described (Marco et al., 1992).
Results Clinical Observations and Mortality Animals in group A did not show clinical signs. In group B, one animal showed lethargy and prostration on day 8 after the first infective dose and two animals showed circling movements on days 8 to 10; one of them was found dead on day 11. In group C, two animals showed circling on day 10 after the first dose, and another two showed incoordination in movement on day 12.
Histopathological and Immunohistochemical Observations Central nervous system. Histological study revealed lesions in two different areas of the CNS (Table 1), namely (1) the brain stem and medulla, and (2) the ventricles. Four mice showed lesions mainly of the brain stem and medulla oblongata (rhombencephalitis), and to a lesser extent the adjacent meninges (Fig. 1). These lesions consisted of mixed inflammatory cellular infiltrates, mainly composed of neutrophils, lymphocytes and macrophages in varying numbers. The cells were distributed perivascularly or formed discrete focal aggregates in the neuropil; they were also associated with vascular structures in the pia mater. The neuropil adjacent to the infiltrates showed marked vacuolation and, in some areas, varying degrees of degeneration and necrosis. In such areas, gliosis and perivascular cuffing could also be detected. Two of the animals with rhombencephalitis had shown circling and one of them was later found dead. One of the group C animals which had shown incoordination in movement showed severe inflammation of the ventricles (Fig. 2), aqueduct (Fig. 3) and meninges (choriomeningitis) on histopathological examination. The inflammatory changes were suppurative, and in some areas the ventricular lumina were completely obliterated (Fig. 4). The infiltrate was associated with foci of degeneration, necrosis and loss of ependymal cells. Inflammatory cells in these foci infiltrated the nervous tissue. In one of the animals which had shown lethargy and prostration, histological examination demonstrated both severe choriomeningitis and a moderate degree of rhombencephalitis. Immunohistochemically, the ABC technique demonstrated moderate to large amounts of L. monocytogenes antigen within the cytoplasm of macrophages and neutrophils, regardless of the area in which the lesions occurred. Other organs. In the liver, three animals in group A and one in group C showed scattered inflammatory foci, containing numerous neutropils and
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Fig. 1.
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Group B mouse, 7 days after first infective dose. Rhombencephalus, showing scattered infiltrates of inflammatory cells in the neuropil. Note absence of ventricular involvement. HE. ×24.
Table 1 Histopathological observations Days after first infective dose
Lesions in mice of group O
A
B
C
4 7 10
None None ···
None None None
13 16
··· ···
··· ···
··· RE + +∗ RE + +∗ RE + and CH + + +∗ None ···
··· ··· RE + +∗ RE +∗ None CH + + +∗
RE, rhombencephalitis; CH, choriomeningitis. +, Mild; + +, moderate; + + +, severe. ∗ One mouse in a subgroup of four. ···, No entry.
macrophages. Small amounts of L. monocytogenes antigen were detected immunohistochemically within phagocytic cells. No histopathological lesions or immunolabelling occurred in any of the other organs examined. Discussion In this murine model, the repeated oral intake of sublethal doses of L. monocytogenes caused CNS lesions similar to those described in man, but no
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Fig. 2.
Group B mouse, 10 days after first infective dose. Severe suppurative inflammation of the choroidal epithelium (arrowheads) and massive inflammatory exudate in the third ventricle. HE. ×182.
Fig. 3.
Group B mouse, 10 days after first infective dose. Complete obliteration of the aqueduct by inflammatory cells, and multifocal rhombencephalitis, in an animal with a mixed distribution pattern of L. monocytogenes-associated lesions. HE. ×91.
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Fig. 4.
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Group C mouse, 16 days after first infective dose. Severe bilateral ventriculitis with focal destruction of the ependymal lining and adjacent neuropil. HE. ×37.
gastrointestinal disease was observed clinically or histopathologically. Gastroenteritis and fever were recently reported in a human outbreak of listeriosis resulting from the consumption of dairy products, the estimated dose of L. monocytogenes being 2·9×1011 (Dalton et al., 1997); it was suggested that a high dose was required to cause gastrointestinal illness. Other workers have shown that only in non-conventional animals (e.g., germ-free or gnotobiotic mice or rats) can enteritis be reproduced by sublethal doses of the order of that used in the present study ( Zachar and Savage, 1979; Czuprynski and Balish, 1981; Okamoto et al., 1984). High doses of L. monocytogenes, such as those used in the present study, have been shown previously to produce systemic subclinical infection by oral or intragastric inoculation (Czuprynski and Balish, 1981; Okamoto et al., 1984; Mano et al., 1992, 1997), but no CNS lesions were reported. Farber and Peterkin (1991) observed that doses as high as 109 organisms were needed to cause a self-limiting septicaemia-like illness in monkeys. We speculate that in the present study the repeated dosing caused a persistent subclinical bacteraemia and the development of CNS listeriosis without gastrointestinal symptoms or generalized disease. This accords with other studies, in which persistent bacteraemia was found to be required for CNS lesions to occur in murine listeriosis (Berche, 1995). The proportion (25%) of animals showing CNS lesions in groups B and C was consistent with recent observations in human adults (McLauchlin, 1990; Farber and Peterkin, 1991; Schuchat et al., 1991) and supports the potential value of our model in the study of CNS lesions in human listeriosis.
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Two histological types of CNS listeriosis were observed in our murine model, namely rhombencephalitis and choriomeningitis. Two animals showed features of both types. The mechanisms by which these two types of lesion develop may be different. Choriomeningitis may be a consequence of normal migration of blood monocytes to the ventricular spaces. Immunohistochemical studies have detected macrophages in the choroid plexus of healthy mice (Gordon et al., 1992). It therefore seems possible that listeria-laden monocytes circulating in the bloodstream reach the vessels and the interstitial tissue of the choroid plexus, leading to inflammation of the ventricles and neighbouring tissues. Studies on the pathogenesis of Streptococcus suis meningitis in pigs showed that circulating monocytes containing phagocytized bacteria migrated into the cerebrospinal fluid (CSF), the choroid plexus acting as a major site of cellular ingress (Williams and Blakemore, 1990). Lysis of listeria-laden macrophages in the choroid plexus attracts other leucocytes (mainly neutrophils), which penetrate the epithelium and accumulate in the CSF (Ta¨uber and Sande, 1984; Leist et al., 1988). These cells may compress and destroy the neighbouring tissues or be carried by the natural flow of CSF through the aqueduct and reach the cisterna magna and the meninges. Narrowing of the aqueduct or ependymal canal may cause parenchymal destruction. The meninges may become affected as a consequence of the circulation of listeria-laden leucocytes in the CSF or directly through vascular irrigation of the meninges at the arachnoidal villi. The pathogenesis of listerial rhombencephalitis in mice is still poorly understood. It has recently been reproduced by intravenous infection (Berche, 1995) or middle ear inoculation (Blanot et al., 1997). In our opinion, these methods do not mimic natural infection routes in human listeriosis. In the present study, the presence of bilateral rhombencephalitis after oral infection, without meningeal or ventricular lesions, indicates that the rhombencephalus may be a target region for L. monocytogenes circulating in the bloodstream (Berche, 1995). In addition, however, neurotropic ascent of the bacteria via cranial nerves has to be considered as a possible invasion route in mice and in man, as has already been demonstrated in ruminants (Charlton and Garcı´a, 1977). In view of the lesions observed, the methods described would seem to form the basis of a valid model for the study of predisposing factors and therapy in human CNS listeriosis. Acknowledgments This work was supported by the Comisio´n Interdepartamental de Ciencia y Tecnologı´a (CICYT), AGF93-C02-02.
References Armstrong, R. W. and Fung, P. C. (1993). Brainstem encephalitis (rhombencephalitis) due to Listeria monocytogenes: case report and review. Clinical Infectious Diseases, 16, 689–702. Bach, M. C. and Davis, K. M. (1987). Listeria rhombencephalitis mimicking tuberculous meningitis. Reviews of Infectious Diseases, 9, 130–133.
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Berche, P. (1995). Bacteremia is required for invasion of the murine central nervous system by Listeria monocytogenes. Microbial Pathogenesis, 18, 323–336. Blanot, S., Joly, M. M., Vilde, F., Jaubert, F., Clement, O., Frija, G. and Berche, P. (1997). A gerbil model for rhombencephalitis due to Listeria monocytogenes. Microbial Pathogenesis, 23, 39–48. Charlton, K. M. and Garcı´a, M. M. (1977). Spontaneous listeric encephalitis and neuritis in sheep. Light microscopy studies. Veterinary Pathology, 14, 297–313. Czuprynski, C. J. and Balish, E. (1981). Pathogenesis of Listeria monocytogenes for gnotobiotic rats. Infection and Immunity, 32, 323–331. Dalton, C. B., Austin, C. C., Sobel, J., Hayes, P. S., Bibb, W. F., Graves, L. M., Swaminathan, B., Proctor, M. E. and Griffin, P. M. (1997). An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk. New England Journal of Medicine, 336, 100–105. Farber, J. M. and Peterkin, P. I. (1991). Listeria monocytogenes, a food-borne pathogen. Microbiological Reviews, 55, 476–511. Gordon, S., Lawson, L., Rabinowitz, S., Crocker, P. R., Morris, L. and Perry, V. H. (1992). Antigen markers of macrophage differentiation in murine tissues. Current Topics in Microbiology and Immunology, 181, 1–37. Ho, J. L., Shands, K. N., Friedland, G., Eckind, P. and Fraser, D. W. (1986). An outbreak of type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals. Archives of Internal Medicine, 146, 520–524. Kirk, J. (1993). Diagnostic ultrastructure of Listeria monocytogenes in human central nervous tissue. Ultrastructural Pathology, 17, 583–592. Leist, T. P., Frei, K., Kam-Hansen, S., Zinkernagel, R. M. and Fontana, A. (1988). Tumor necrosis factor alpha in cerebrospinal fluid during bacterial but not viral meningitis. Journal of Experimental Medicine, 167, 1743–1748. Lorber, B. (1997). Listeriosis. Clinical Infectious Diseases, 24, 1–11. Marco, A. J., Altimira, J., Prats, N., Lo´pez, S., Domı´nguez, L., Domingo, M. and Briones, V. (1997). Penetration of Listeria monocytogenes in mice infected by the oral route. Microbial Pathogenesis, 23, 255–263. Marco, A. J., Prats, N., Ramos, J. A., Briones, V., Blanco, M., Domı´nguez, L. and Domingo, M. (1992). A microbiological, histopathological and immunohistological study of the intragastric inoculation of Listeria monocytogenes in mice. Journal of Comparative Pathology, 107, 1–9. McLauchlin, J. (1990). Human listeriosis in Britain, 1967–85, a summary of 722 cases. Epidemiology and Infection, 104, 191–201. Okamoto, M., Nakane, A. and Minagawa, T. (1984). Host resistance to an intragastric infection with Listeria monocytogenes in mice depends on cellular immunity and intestinal bacterial flora. Infection and Immunity, 62, 3080–3085. Prats, N., Briones, V., Blanco, M. M., Altimira, J., Ramos, J. A., Domı´nguez, L. and Marco, A. J. (1992). Choroiditis and meningitis in experimental murine infection with Listeria monocytogenes. European Journal of Clinical and Microbiological Infectious Diseases, 11, 744–747. Schlech, W. F., Lavigne, P. M., Bortolussi, R. A., Allen, A. C., Haldane, E. V., Wort, A. J., Hightower, A. W., Johnson, S. E., King, S. H., Nicholls, E. S. and Broome, C. V. (1983). Epidemic listeriosis. Evidence for transmission by food. New England Journal of Medicine, 308, 203–206. Schuchat, A., Swaminathan, B. and Broome, C. V. (1991). Epidemiology of human listeriosis. Clinical Microbiology Reviews, 4, 169–183. Skogberg, K., Syrja¨nen, J., Jahkola, M., Renkonen, O. V., Paavonen, J., Ahonen, J., Kontiainen, S., Ruutu, P. and Valtonen, V. (1992). Clinical presentation and outcome of listeriosis in patients with and without immunosuppressive therapy. Clinical Infectious Diseases, 14, 815–821. Ta¨uber, M. G. and Sande, M. A. (1984). Pathogenesis of bacterial meningitis: contributions by experimental models in rabbits. Infection, 12, 3–10.
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Williams, A. E. and Blakemore, W. F. (1990). Pathogenesis of meningitis caused by Streptococcus suis type 2. Journal of Infectious Diseases, 162, 474–481. Yamauchi, J., Namiki, M., Otsubo, O., Akiyama, N. and Inou, T. (1978). Choriomeningo-encephalitis due to Listeria monocytogenes after renal transplantation. Japanese Journal of Experimental Medicine, 48, 143–148. Zachar, Z. and Savage, D. C. (1979). Microbial interference and colonization of the murine gastrointestinal tract by Listeria monocytogenes. Infection and Immunity, 23, 168–174.
Received, July 9th, 1998 Accepted, February 4th, 1999
Repeated Oral Dosing with Listeria monocytogenes in Mice as a Model of Central Nervous System Listeriosis in Man J. Altimira, N. Prats, S. Lo´pez, M. Domingo, V. Briones∗, L. Domı´nguez∗ and A. Marco Departamento de Patologı´a y Produccio´n Animales (Histologı´a y Anatomı´a Patolo´gica), Facultad de Veterinaria, Universidad Auto´noma de Barcelona, 08193 Bellaterra (Barcelona) and ∗Departamento de Patologı´a Animal I, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain Summary Human listeriosis is a food-borne disease of immunosuppressed or previously healthy adults. The repeated oral administration of a sublethal dose (5×109 colony-forming units) of Listeria monocytogenes for 7 or 10 consecutive days led to the development of severe central nervous system (CNS) lesions in 25% of experimental mice. Histopathological examination of the brain revealed rhombencephalitis and ventriculitis as two distinct inflammatory patterns, resembling those seen in human listeriosis. This model would seem to be potentially useful for research on pathogenesis, predisposing factors and therapy in CNS listeriosis in man. 1999 W.B. Saunders Company Limited
Introduction Infection of the central nervous system (CNS) by Listeria monocytogenes is currently considered one of the most common forms of human listeriosis particularly in the newborn and in immunosuppressed patients; it sometimes occurs, however, in previously healthy persons (Ho et al., 1986; McLauchlin, 1990; Farber and Peterkin, 1991). Human listeriosis is regarded as a foodborne disease (Farber and Peterkin, 1991; Schuchat et al., 1991; Lorber, 1997) and food products with an overgrowth of L. monocytogenes have sometimes been incriminated as the source of infection (Schlech et al., 1983; Farber and Peterkin, 1991). However, there is still debate as to why only a small proportion of healthy persons exposed to a contaminated food source develop the disease (Farber and Peterkin, 1991). Three different patterns of inflammatory lesion in the CNS have been described in human listeriosis. Meningitis is the most common form in immunosuppressed and neonatal subjects but is also occasionally reported in previously healthy persons (McLauchlin, 1990; Schuchat et al., 1991; Skogberg et al., 1992). Rhombencephalitis has been reported in adults (Bach and Davis, 1987; Armstrong and Fung, 1993; Kirk, 1993). Choriomeningitis and periventriculitis have also been described, sometimes in Correspondence to: A. Marco. 0021–9975/99/060117+09 $12.00
1999 W.B. Saunders Company Limited
118
J. Altimira et al.
association with septicaemic lesions in organs outside the CNS (Yamauchi et al., 1978). Listerial rhombencephalitis has often been described in adult sheep and cattle and there is evidence that L. monocytogenes passes along cranial nerves and causes encephalitis, mainly affecting the brain stem (Charlton and Garcı´a, 1977). The aim of the present study was to develop an experimental model in which CNS lesions mimicking those seen in the human species were reproduced. In previously described murine models, highly artificial methods of infection (intracranial, middle ear, subcutaneous or intravenous) were used (Leist et al., 1988; Prats et al., 1992; Berche, 1995; Blanot et al., 1997). In our opinion, these methods, with their devastating effects, do not represent an accurate experimental model for natural human listeriosis, in which oral infection is commonly assumed. Outbreaks of human listeriosis sometimes last for weeks or months, suggesting prolonged exposure to the bacteria (Schuchat et al., 1991). It would seem, therefore, that patients may consume a contaminated food (e.g., raw vegetables, soft cheese) for some time before showing clinical signs. To mimic this situation, mice were given daily oral doses for varying periods. In this way it was possible to study CNS lesions produced by multiple sublethal doses of the organism. Materials and Methods Mice Swiss CD1 mice (Panlab, Barcelona) of either sex, aged 6 weeks and weighing 20 g, were used. Four groups (O, A, B and C) were established. Mice of group O (n=6), given a single oral dose of L. monocytogenes, were humanely killed for necropsy in subgroups of three on days 4 and 7 after infection. Mice of groups A, B and C (n=12/group) were dosed orally once each day for 4, 7 and 10 consecutive days, respectively, and killed in subgroups of four for necropsy on days 1, 4 and 7 after the final dose. (The third subgroup of group B consisted of only three mice, because one animal had died.) Bacterial Inocula Strain P-14B of L. monocytogenes serovar 4b, of human origin, was grown on brain–heart infusion agar (BHI; Difco, USA) at 37°C for 22 h. The organisms were centrifuged and resuspended in sterile 0·9% saline. The concentration of the cell suspension was adjusted spectrophotometrically at 550 nm and diluted to obtain the desired daily inoculum of 5×109 colony-forming units of L. monocytogenes; this was administered orally by a feeding bottle as previously described (Marco et al., 1997). Histopathological and Immunohistochemical Examination of Samples Samples of liver, spleen, lymph nodes (mesenteric, maxillary and lumbar), stomach, small and large intestine (several samples from each), kidney, adrenal gland, urinary bladder, reproductive organs, lung, heart, thymus and brain were obtained immediately after euthanasia, fixed for at least 48 h in 10% buffered formalin and embedded in paraffin wax by routine procedures. Serial sections (4 lm) were
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stained with haematoxylin and eosin (HE) or processed for immunohistochemical examination by the avidin–biotin complex (ABC) method, as previously described (Marco et al., 1992).
Results Clinical Observations and Mortality Animals in group A did not show clinical signs. In group B, one animal showed lethargy and prostration on day 8 after the first infective dose and two animals showed circling movements on days 8 to 10; one of them was found dead on day 11. In group C, two animals showed circling on day 10 after the first dose, and another two showed incoordination in movement on day 12.
Histopathological and Immunohistochemical Observations Central nervous system. Histological study revealed lesions in two different areas of the CNS (Table 1), namely (1) the brain stem and medulla, and (2) the ventricles. Four mice showed lesions mainly of the brain stem and medulla oblongata (rhombencephalitis), and to a lesser extent the adjacent meninges (Fig. 1). These lesions consisted of mixed inflammatory cellular infiltrates, mainly composed of neutrophils, lymphocytes and macrophages in varying numbers. The cells were distributed perivascularly or formed discrete focal aggregates in the neuropil; they were also associated with vascular structures in the pia mater. The neuropil adjacent to the infiltrates showed marked vacuolation and, in some areas, varying degrees of degeneration and necrosis. In such areas, gliosis and perivascular cuffing could also be detected. Two of the animals with rhombencephalitis had shown circling and one of them was later found dead. One of the group C animals which had shown incoordination in movement showed severe inflammation of the ventricles (Fig. 2), aqueduct (Fig. 3) and meninges (choriomeningitis) on histopathological examination. The inflammatory changes were suppurative, and in some areas the ventricular lumina were completely obliterated (Fig. 4). The infiltrate was associated with foci of degeneration, necrosis and loss of ependymal cells. Inflammatory cells in these foci infiltrated the nervous tissue. In one of the animals which had shown lethargy and prostration, histological examination demonstrated both severe choriomeningitis and a moderate degree of rhombencephalitis. Immunohistochemically, the ABC technique demonstrated moderate to large amounts of L. monocytogenes antigen within the cytoplasm of macrophages and neutrophils, regardless of the area in which the lesions occurred. Other organs. In the liver, three animals in group A and one in group C showed scattered inflammatory foci, containing numerous neutropils and
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Fig. 1.
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Group B mouse, 7 days after first infective dose. Rhombencephalus, showing scattered infiltrates of inflammatory cells in the neuropil. Note absence of ventricular involvement. HE. ×24.
Table 1 Histopathological observations Days after first infective dose
Lesions in mice of group O
A
B
C
4 7 10
None None ···
None None None
13 16
··· ···
··· ···
··· RE + +∗ RE + +∗ RE + and CH + + +∗ None ···
··· ··· RE + +∗ RE +∗ None CH + + +∗
RE, rhombencephalitis; CH, choriomeningitis. +, Mild; + +, moderate; + + +, severe. ∗ One mouse in a subgroup of four. ···, No entry.
macrophages. Small amounts of L. monocytogenes antigen were detected immunohistochemically within phagocytic cells. No histopathological lesions or immunolabelling occurred in any of the other organs examined. Discussion In this murine model, the repeated oral intake of sublethal doses of L. monocytogenes caused CNS lesions similar to those described in man, but no
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Fig. 2.
Group B mouse, 10 days after first infective dose. Severe suppurative inflammation of the choroidal epithelium (arrowheads) and massive inflammatory exudate in the third ventricle. HE. ×182.
Fig. 3.
Group B mouse, 10 days after first infective dose. Complete obliteration of the aqueduct by inflammatory cells, and multifocal rhombencephalitis, in an animal with a mixed distribution pattern of L. monocytogenes-associated lesions. HE. ×91.
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Fig. 4.
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Group C mouse, 16 days after first infective dose. Severe bilateral ventriculitis with focal destruction of the ependymal lining and adjacent neuropil. HE. ×37.
gastrointestinal disease was observed clinically or histopathologically. Gastroenteritis and fever were recently reported in a human outbreak of listeriosis resulting from the consumption of dairy products, the estimated dose of L. monocytogenes being 2·9×1011 (Dalton et al., 1997); it was suggested that a high dose was required to cause gastrointestinal illness. Other workers have shown that only in non-conventional animals (e.g., germ-free or gnotobiotic mice or rats) can enteritis be reproduced by sublethal doses of the order of that used in the present study ( Zachar and Savage, 1979; Czuprynski and Balish, 1981; Okamoto et al., 1984). High doses of L. monocytogenes, such as those used in the present study, have been shown previously to produce systemic subclinical infection by oral or intragastric inoculation (Czuprynski and Balish, 1981; Okamoto et al., 1984; Mano et al., 1992, 1997), but no CNS lesions were reported. Farber and Peterkin (1991) observed that doses as high as 109 organisms were needed to cause a self-limiting septicaemia-like illness in monkeys. We speculate that in the present study the repeated dosing caused a persistent subclinical bacteraemia and the development of CNS listeriosis without gastrointestinal symptoms or generalized disease. This accords with other studies, in which persistent bacteraemia was found to be required for CNS lesions to occur in murine listeriosis (Berche, 1995). The proportion (25%) of animals showing CNS lesions in groups B and C was consistent with recent observations in human adults (McLauchlin, 1990; Farber and Peterkin, 1991; Schuchat et al., 1991) and supports the potential value of our model in the study of CNS lesions in human listeriosis.
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Two histological types of CNS listeriosis were observed in our murine model, namely rhombencephalitis and choriomeningitis. Two animals showed features of both types. The mechanisms by which these two types of lesion develop may be different. Choriomeningitis may be a consequence of normal migration of blood monocytes to the ventricular spaces. Immunohistochemical studies have detected macrophages in the choroid plexus of healthy mice (Gordon et al., 1992). It therefore seems possible that listeria-laden monocytes circulating in the bloodstream reach the vessels and the interstitial tissue of the choroid plexus, leading to inflammation of the ventricles and neighbouring tissues. Studies on the pathogenesis of Streptococcus suis meningitis in pigs showed that circulating monocytes containing phagocytized bacteria migrated into the cerebrospinal fluid (CSF), the choroid plexus acting as a major site of cellular ingress (Williams and Blakemore, 1990). Lysis of listeria-laden macrophages in the choroid plexus attracts other leucocytes (mainly neutrophils), which penetrate the epithelium and accumulate in the CSF (Ta¨uber and Sande, 1984; Leist et al., 1988). These cells may compress and destroy the neighbouring tissues or be carried by the natural flow of CSF through the aqueduct and reach the cisterna magna and the meninges. Narrowing of the aqueduct or ependymal canal may cause parenchymal destruction. The meninges may become affected as a consequence of the circulation of listeria-laden leucocytes in the CSF or directly through vascular irrigation of the meninges at the arachnoidal villi. The pathogenesis of listerial rhombencephalitis in mice is still poorly understood. It has recently been reproduced by intravenous infection (Berche, 1995) or middle ear inoculation (Blanot et al., 1997). In our opinion, these methods do not mimic natural infection routes in human listeriosis. In the present study, the presence of bilateral rhombencephalitis after oral infection, without meningeal or ventricular lesions, indicates that the rhombencephalus may be a target region for L. monocytogenes circulating in the bloodstream (Berche, 1995). In addition, however, neurotropic ascent of the bacteria via cranial nerves has to be considered as a possible invasion route in mice and in man, as has already been demonstrated in ruminants (Charlton and Garcı´a, 1977). In view of the lesions observed, the methods described would seem to form the basis of a valid model for the study of predisposing factors and therapy in human CNS listeriosis. Acknowledgments This work was supported by the Comisio´n Interdepartamental de Ciencia y Tecnologı´a (CICYT), AGF93-C02-02.
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Received, July 9th, 1998 Accepted, February 4th, 1999