744
15. Hadziyannis SJ, Lieberman H, Karvountzis G, Shafritz D: Analysis of liver disease, nuclear HBcAg, viral replication and hepatitis B virus DNA in liver and serum of HBeAg vs anti-HBe positive carriers of hepatitis B virus. Hepatology 1983, 3: 656--662. 16. Brechot C, Degos F, Lugassy C, Thiers V, Zafrani S,
France D, Bismuth H, Trepo C, Benhamou JP, Wands J, lsselbacher K, Tiollais P, Berthelot P: Hepatitis B virus DNA in patients with chronic liver disease and negative tests for hepatitis B surface antigen. New England Journal of Medicine 1985, 312: 270-276. 17. Shafritz DA, Lieberman HM, Isselbacher K, Wands
J: Monoclonal radioimmunoassayfor hepatitis B surface antigen: demonstration of hepatitis B virus DNA or related sequences in serum and viral epitopes in immune complexes. Proceedings of the National Academy of Science of the USA 1982, 79: 5675-5679.
Choroiditis and Meningitis in Experimental Murine Infection with Listeria monocytogenes N. Prats 2, V. Briones 1, M.M. Blanco 1, J. Altimira 2, J.A. R a m o s 2, L. D o m f n g u e z 1, A. M a r c o 2.
In a study of central nervous system involvement in experimental listeriosis 27 Swiss CD1 mice were inoculated subcutaneously with Listeria monocytogenes. Systemic infection developed, as shown by the isolation ofListeria monocytogenes and histopathological lesions in the spleen and liver. In the central nervous system a mixed inflammatory infiltration in the ventricular system, especially in the choroid plexus, and leptomeningifts were the most relevant lesions. Inflammatory lesions were associated with the presence of Llsteria monocytogenes, as demonstrated by a positive anti-Listeria monocytogenes immunoperoxidase reaction within phagocytic cells. It is suggested that choroiditis and meningitis developed as a consequence of hematogenous dissemination of Listeria monoeytogenes within
1Departamento de Patologfa Animal I, Faeultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. 2Departamento de Patolog[a y Producciones Animales (Anatomfa Patol6gica), Facultad de Veterinaria Universidad Aut6noma de Barcelona, 08193 (Bellaterra) Barcelona, Spain.
Eur. J. Clin. Microbiol. Infect. Dis.
mononuclear phagocytes and penetration of these cells into the ventricular system through the choroid plexus.
There are two main forms of nervous system involvement in listeriosis: encephalitis, which is the predominant form in small ruminants (1), and meningitis, which is the most frequent form of listeriosis in humans (2). In listeric encephalitis in small ruminants, involvement of the medulla oblongata and pens is a consequence of neurotropic ascension of Listeria monocytogenes via the trigeminal nerve (1), whereas in cases of meningitis in humans and other species, spread of the microorganism to the central nervous system (CNS) is assumed to be hematogenous. Nervous system involvement in murine listeriosis has also been reported (3), but a clear-cut distinction between encephalitis and meningitis has not been established in this experimental model. Moreover, most of the studies on listeriosis in mice report no changes in the CNS (4-6). In this paper we report on an experimental model of CNS infection in mice infected subcutaneously with Listeria monocytogenes, which is similar to the meningitic form in humans. Materials and Methods. Listeria monocytogenes (type strain, serovar 1/2a, SLCC 2371/NCTC 7973) was grown in 250 ml of brain heart infusion broth (BHI; Difco, USA) at 37 °C for 22 h, centrifuged and resuspended in 20 ml of sterile 0.9 % saline. This suspension (2 x 101° cfu/ml) was diluted to obtain the desired inoculum. Twenty-seven 6-week-old female Swiss CD1 listeria-resistant mice (Interfauna, Spain) were inoculated subcutaneously with 1 x 109 cfu/ml of Listeria monocytogenes. This dose represents approximately 1.3 x LD50 for this route and for this strain of mice (7). Three animals were sacrificed daily from day 1 to day 9 post-inoculation by inhalation of ethyl ether. The mice to be sacrificed were randomly selected. Necropsy was performed under aseptic conditions. Samples of the liver and spleen were weighed and homogenated in 0.9 % saline, and serial dilutions plated in duplicate on BHI agar for the counting of Listeria monocytogenes. The whole brain, and samples from the liver and spleen were removed, fixed in 10 % formol saline and embedded in paraffin according to routine histological procedures. Sections were stained with hematoxylin and eosin (HE) or processed for immunocytochemistry by the peroxidase anti-
Vo1.11,1992
745
p e r o x i d a s e t e c h n i q u e ( P A P ) t o d e t e c t Listeria monocytogenes a n t i g e n , a s p r e v i o u s l y d e s c r i b e d
between day 2 and day 9 post-inoculation. Bact e r i a l c o u n t s in t h e l i v e r a n d s p l e e n i n c r e a s e d from day 1 post-inoculation on, reaching the highest values on days 3-5 post-inoculation and gradually decreasing until day 9 post-inoculation.
(6). B r i e f l y , s l i d e s w e r e i n c u b a t e d f o r 30 m i n e a c h in s p e c i f i c anti-Listeria monocytogenes s e r u m ( d i l u t e d 1:2000), g o a t - a n t i r a b b i t IgG serum (1:160) a n d r a b b i t P A P c o m p l e x (1:200), a n d r i n s e d in Tris b u f f e r e d s a l i n e a f t e r e a c h i n c u b a t i o n . D i a m i n o b e n z i d i n e ( D A B ) a n d 0.5 % H 2 0 2 i n i m i d a z o l - b u f f e r ( p H 7.2, 0.1 M ) w e r e u s e d as chromogen and substrate for peroxidase.
T h e m o s t r e l e v a n t h i s t o p a t h o l o g i c a l f i n d i n g in the CNS was a mixed inflammatory infiltrate (polymorphonuclear neutrophils and monon u c l e a r p h a g o c y t e s ) a f f e c t i n g t h e v e n t r i c u l a r system and meninges. Lesions involving the ventricular system were especially marked in the c h o r o i d p l e x u s , a n d w e r e n o t i c e a b l e in t h e l a t e r a l ventricles, third ventricle, mesencephalic aqueduct, fourth ventricle and central canal of the s p i n a l c o r d ( F i g u r e 1). A p o s i t i v e P A P r e a c t i o n w a s o b s e r v e d in Listeria monocytogenes-laden macrophages and neutrophils associated with the l e s i o n s ( T a b l e 1).
Results and Discussion. N e u r o l o g i c a l c h a n g e s w e r e first o b s e r v e d o n d a y 6 p o s t - i n o c u l a t i o n , w h e n 7 o f 12 m i c e s h o w e d p a r e s i a o f t h e h i n d limbs. In the period from day 7 to day 9 post-inoculation, many mice showed ataxia and two mice circling movements. Three mice died on day 5 p o s t - i n o c u l a t i o n , a n d t w o d i e d d a i l y in t h e p e r i o d from day 6 to day 9 post-inoculation. The course o f i n f e c t i o n is s u m m a r i z e d in T a b l e 1. C h a r a c t e r i s t i c h e p a t i c o r s p l e n i c l e s i o n s as d e s c r i b e d b y o t h e r a u t h o r s (5, 6, 8) w e r e o b s e r v e d in t h e p e r i o d
Our results show that inflammation of the ventricular system (especially the choroid plexus) and meninges, with the presence of intracellular
Table 1: Intensity of inflammatory lesions, immunoperoxidase reaction and bacterial counts in mice inoculated sub-
cutaneously with 1 x 10 9 cfu of Listeria monocytogenes. Day
Mouse no.
Ventricles PAP
HE
1
2 3
4 5
6
7
8
9
1 2 3 4 5 6 7 8 9 10 11 12 13" 14" 15" 16" 17" 18 19 20* 21" 22 23* 24* 25* 26* 27
Meninges
Parenchyma
Liver
Spleen
--
. . . . . . . . . + + . + + + +++ +++ . ++ +++ +++ . +++ +++ +++ +++ .
. . . . . . . . .
HE . . .
. . . . . . + +
.
. + + + ++ ++
.
. + +++ +++
.
. +++ +++ +++ +++
.
.
. . .
PAP . . .
HE . . .
. . . . . . . . . . . . . . . . . . + + . . . . . . . + + . . . . + + + + + + . . . + + ++ ++ ++ ++ . . . ++ ++ ++ ++ + + + + . . .
PAP . . .
PAP
. . .
+
+
-
-
-
-
+ -
+ -
+
+ .
HE
+/+ + + + + ++ + + + + ++ ++ ++ ++ ++ ++ + + ++ + + + .
+ + +/+/+ +/+ + + ++ ++ ++ + + + + ++ + +/-
Log cfutg 3.70 3.66 3.61 5.20 5.65 5.42 5.81 5.69 5.84 6.11 5.91 5.83 7.43 ND 7.26 6.01 5.65 5.70 5.12 ND 5.21 3.30 2.94 ND ND 2.81 2.89
HE + + ++ ++ ++ ++ +++ ++ +++ +++ +++ +++ +++ ++ ++ + +++ +++ +++ ++ + + -
PAP + + + + + + + + + +++ ++ ++ + + + +/+ + + + -
Log efulg 4.95 4.78 5.01 7.21 6.96 7.18 7.70 7.90 7.58 8.20 7.95 7.74 8.83 ND 9.21 7.20 6.60 6.27 6.05 ND 5.93 3.34 3.03 ND ND 2.24 2.46
HE: hematoxylin-eosin; PAP: peroxidase-antiperoxidase -: no lesion (HE)/no reaction (PAP); +/-: minimal lesion/reaction; +: low grade lesion/reaction; ++: moderate lesion/reaction; +++: intense lesion/reaction; ND: not done (autolytie); *: died.
746
Eur. J. Clin. Microbiol. Infect. Dis.
A
B
Figure 1: A: Third ventricle of a Listeria monocytogenes-infected mouse (day 6 post-inoculation) showing mild choroiditis. Note inflammatory cells in choroid plexus and in lumen. B: Lateral ventricle of a Listeria monocytogenes-infeeted mouse (day 9 post-inoculation). A profuse exudation of inflammatory cells into the ventricular space and necrosis of the choroidal epithelial cells (arrow) can be seen (Bar = 50 •m).
Listeria monocytogenes within phagocytic cells, is the most relevant pathological finding in the CNS after subcutaneous inoculation in mice. Encephalitis in this murine model was not as frequent and intense as described in the literature (3). Inflammatory lesions of the ports and medulla oblongata, as described in the rhombencephalitic form of small-ruminant listeriosis, were not observed. The incidence and severity of neurological symptoms and lesions increased when the bacterial counts in the liver and spleen began to decline, showing that CNS involvement is a late
phenomenon in septicemic listeriosis. Using the subcutaneous route, the course of the infection, as determined from peak multiplication of Listeria monocytogenes in the liver and spleen, is slightly retarded compared to infection after intravenous or intraperitoneal inoculation (4, 5, 7). This fact might explain the high rate of CNS symptoms and lesions seen in our experiments. The choroid plexus plays an essential role in the development of meningitis in other experimental models of bacterial infection (9, 10). Choroiditis is the first and most constant lesion produced by
Vol. 11, 1992
Streptococcus suis type 2 in CNS (10), and intracellular Streptococcus suis type 2 organisms are always associated with the lesions. T h e s e investigations indicated that the choroid plexus is the site at which Streptococcus suis type 2 within m o n o n u c l e a r phagocytes gains access to cerebrospinal fluid (CSF) spaces, O u r data suggest that Listeria rnonocytogenes also reaches the choroid plexus within m o n o n u c l e a r phagocytes, probably along the normal migration routes o f these ceils (11), causing choroiditis, ependymitis, meningitis and finally encephalitis. T h e intensity of the inflammatory lesions in the choroid plexus is presumably greater than in other ependymal structures whereas meningitis is of moderate severity, and encephalitis is infrequent and much less severe. O t h e r data support these assumptions: 1) the presence of free Listeria monocytogenes organisms in the bloodstream is known to be only a transient phase in experimental listeriosis (4, 5); 2) most organisms (90 % ) are captured and killed after intravenous inoculation by macrophages resident in the liver and spleen (5, 8); 3) surviving Listeria monocytogenes begin a rapid multiplication within the resident or inflammatory macrophages (5, 12); 4) Listeria monocytogenes-laden m o n o n u c l e a r phagocytes transport bacteria throughout the host organism, especially to sites, such as lymphoid organs (13), in which the presence of mononuclear phagocytes is a normal feature. In the CNS, a population of m o n o n u c l e a r phagocytes can be found associated with the epithelium of the ventricular system, particularly, the epithelium of the choroid plexus (11). The presence of such cells underlying the ependymal epithelium strongly suggests that they may play a role in CNS involvement during systemic listeriosis. In conclusion, our findings are in agreement with the hypothesis that mononuclear phagocytes and the choroid plexus play a substantial role in the pathogenic mechanism of some bacterial meningitides (10). Furthermore, choroiditis and meningitis can be r e p r o d u c e d by subcutaneous inoculation in the Swiss CD1 strain of mice, this being in
747
our opinion a suitable model for listeric meningitis in humans and other species.
References
1. Charlton KM, Garcla MM: Spontaneous listeric encephalitis and neuritis in sheep. Light microscopy studies. Veterinary Pathology 1977, 14: 297-313. 2. Lorber B: Clinical listeriosis implications for pathogenesis. In: Miller A J, Smith JL, Somkuti GA (ed): Foodborne listeriosis. Amsterdam, Elsevier, 1990, p. 41-49. 3. Cordy DR, Osebold JW: The neuropathogenesis of Listeria encephalomyelitis in sheep and mice. Journal of Infectious Diseases 1959, 104: 164-173. 4. Mackaness GB: Cellular resistance to infection. Journal of Experimental Medicine 1962, 116: 381-406. 5. Mandel TE, Cheers C: Resistance and susceptibility of mice to bacterial infection: histopathology of listeriosis in resistant and susceptible strains. Infection and Immunity 1980, 30: 851--861. 6. Donfingo M, Ramos JA, Domlnguez L, Ferrer L, Marco A: Demonstration of Listeria monocytogenes with the PAP technique in formalin fixed paraffin embedded tissues of experimentally infected mice. Journal of Veterinary Medicine 1986, 33: 537-542. 7. Briones V, Blanco MM, Marco A, Prats N, Fern~ndezGaray~bal JF, Su~rez G, Domingo M, Dominguez L: Biliar excretion of Listeria monocytogenes as origin of fecal carriers. American Journal of Veterinary Research 1992, 53: 191-193. 8. North RJ:The relative importance of blood monocytes and fixed macrophages to the expression of cellmediated immunity to infection. Journal of Experimental Medicine 1970, 132: 521-534. 9. Daum RS, Scheifele DW, Syriopoulou VP, Averill D, Smith AL: Ventricular involvement in experimental Haemophilus h~fluenzae meningitis. Journal of Pediatrics 1978, 93: 927-930. 10. Williams AE, Blakemore WF: Pathogenesis of meningitis caused by Streptococcus suis type 2. Journal of Infectious Diseases t990, 162: 474-481. 11. Oehmichen M: Are resting and/or reactive mieroglia macrophages? Immunobiology 1982, 161: 246-254. 12. Berche P, Galliard JL, Richard S: Invasiveness and intracellular growth of Listeria monocytogenes. Infection 1988, 16, Supplement 2: 145-148. 13. Marco A, Domingo M, Prats N, Briones V, Pumarola M, Domfnguez L: Pathogenesis of lymphoid lesions in murine experimental listeriosis. Journal of Comparative Pathology 1991, 105: 1-15.
15. Hadziyannis SJ, Lieberman H, Karvountzis G, Shafritz D: Analysis of liver disease, nuclear HBcAg, viral replication and hepatitis B virus DNA in liver and serum of HBeAg vs anti-HBe positive carriers of hepatitis B virus. Hepatology 1983, 3: 656--662. 16. Brechot C, Degos F, Lugassy C, Thiers V, Zafrani S,
France D, Bismuth H, Trepo C, Benhamou JP, Wands J, lsselbacher K, Tiollais P, Berthelot P: Hepatitis B virus DNA in patients with chronic liver disease and negative tests for hepatitis B surface antigen. New England Journal of Medicine 1985, 312: 270-276. 17. Shafritz DA, Lieberman HM, Isselbacher K, Wands
J: Monoclonal radioimmunoassayfor hepatitis B surface antigen: demonstration of hepatitis B virus DNA or related sequences in serum and viral epitopes in immune complexes. Proceedings of the National Academy of Science of the USA 1982, 79: 5675-5679.
Choroiditis and Meningitis in Experimental Murine Infection with Listeria monocytogenes N. Prats 2, V. Briones 1, M.M. Blanco 1, J. Altimira 2, J.A. R a m o s 2, L. D o m f n g u e z 1, A. M a r c o 2.
In a study of central nervous system involvement in experimental listeriosis 27 Swiss CD1 mice were inoculated subcutaneously with Listeria monocytogenes. Systemic infection developed, as shown by the isolation ofListeria monocytogenes and histopathological lesions in the spleen and liver. In the central nervous system a mixed inflammatory infiltration in the ventricular system, especially in the choroid plexus, and leptomeningifts were the most relevant lesions. Inflammatory lesions were associated with the presence of Llsteria monocytogenes, as demonstrated by a positive anti-Listeria monocytogenes immunoperoxidase reaction within phagocytic cells. It is suggested that choroiditis and meningitis developed as a consequence of hematogenous dissemination of Listeria monoeytogenes within
1Departamento de Patologfa Animal I, Faeultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. 2Departamento de Patolog[a y Producciones Animales (Anatomfa Patol6gica), Facultad de Veterinaria Universidad Aut6noma de Barcelona, 08193 (Bellaterra) Barcelona, Spain.
Eur. J. Clin. Microbiol. Infect. Dis.
mononuclear phagocytes and penetration of these cells into the ventricular system through the choroid plexus.
There are two main forms of nervous system involvement in listeriosis: encephalitis, which is the predominant form in small ruminants (1), and meningitis, which is the most frequent form of listeriosis in humans (2). In listeric encephalitis in small ruminants, involvement of the medulla oblongata and pens is a consequence of neurotropic ascension of Listeria monocytogenes via the trigeminal nerve (1), whereas in cases of meningitis in humans and other species, spread of the microorganism to the central nervous system (CNS) is assumed to be hematogenous. Nervous system involvement in murine listeriosis has also been reported (3), but a clear-cut distinction between encephalitis and meningitis has not been established in this experimental model. Moreover, most of the studies on listeriosis in mice report no changes in the CNS (4-6). In this paper we report on an experimental model of CNS infection in mice infected subcutaneously with Listeria monocytogenes, which is similar to the meningitic form in humans. Materials and Methods. Listeria monocytogenes (type strain, serovar 1/2a, SLCC 2371/NCTC 7973) was grown in 250 ml of brain heart infusion broth (BHI; Difco, USA) at 37 °C for 22 h, centrifuged and resuspended in 20 ml of sterile 0.9 % saline. This suspension (2 x 101° cfu/ml) was diluted to obtain the desired inoculum. Twenty-seven 6-week-old female Swiss CD1 listeria-resistant mice (Interfauna, Spain) were inoculated subcutaneously with 1 x 109 cfu/ml of Listeria monocytogenes. This dose represents approximately 1.3 x LD50 for this route and for this strain of mice (7). Three animals were sacrificed daily from day 1 to day 9 post-inoculation by inhalation of ethyl ether. The mice to be sacrificed were randomly selected. Necropsy was performed under aseptic conditions. Samples of the liver and spleen were weighed and homogenated in 0.9 % saline, and serial dilutions plated in duplicate on BHI agar for the counting of Listeria monocytogenes. The whole brain, and samples from the liver and spleen were removed, fixed in 10 % formol saline and embedded in paraffin according to routine histological procedures. Sections were stained with hematoxylin and eosin (HE) or processed for immunocytochemistry by the peroxidase anti-
Vo1.11,1992
745
p e r o x i d a s e t e c h n i q u e ( P A P ) t o d e t e c t Listeria monocytogenes a n t i g e n , a s p r e v i o u s l y d e s c r i b e d
between day 2 and day 9 post-inoculation. Bact e r i a l c o u n t s in t h e l i v e r a n d s p l e e n i n c r e a s e d from day 1 post-inoculation on, reaching the highest values on days 3-5 post-inoculation and gradually decreasing until day 9 post-inoculation.
(6). B r i e f l y , s l i d e s w e r e i n c u b a t e d f o r 30 m i n e a c h in s p e c i f i c anti-Listeria monocytogenes s e r u m ( d i l u t e d 1:2000), g o a t - a n t i r a b b i t IgG serum (1:160) a n d r a b b i t P A P c o m p l e x (1:200), a n d r i n s e d in Tris b u f f e r e d s a l i n e a f t e r e a c h i n c u b a t i o n . D i a m i n o b e n z i d i n e ( D A B ) a n d 0.5 % H 2 0 2 i n i m i d a z o l - b u f f e r ( p H 7.2, 0.1 M ) w e r e u s e d as chromogen and substrate for peroxidase.
T h e m o s t r e l e v a n t h i s t o p a t h o l o g i c a l f i n d i n g in the CNS was a mixed inflammatory infiltrate (polymorphonuclear neutrophils and monon u c l e a r p h a g o c y t e s ) a f f e c t i n g t h e v e n t r i c u l a r system and meninges. Lesions involving the ventricular system were especially marked in the c h o r o i d p l e x u s , a n d w e r e n o t i c e a b l e in t h e l a t e r a l ventricles, third ventricle, mesencephalic aqueduct, fourth ventricle and central canal of the s p i n a l c o r d ( F i g u r e 1). A p o s i t i v e P A P r e a c t i o n w a s o b s e r v e d in Listeria monocytogenes-laden macrophages and neutrophils associated with the l e s i o n s ( T a b l e 1).
Results and Discussion. N e u r o l o g i c a l c h a n g e s w e r e first o b s e r v e d o n d a y 6 p o s t - i n o c u l a t i o n , w h e n 7 o f 12 m i c e s h o w e d p a r e s i a o f t h e h i n d limbs. In the period from day 7 to day 9 post-inoculation, many mice showed ataxia and two mice circling movements. Three mice died on day 5 p o s t - i n o c u l a t i o n , a n d t w o d i e d d a i l y in t h e p e r i o d from day 6 to day 9 post-inoculation. The course o f i n f e c t i o n is s u m m a r i z e d in T a b l e 1. C h a r a c t e r i s t i c h e p a t i c o r s p l e n i c l e s i o n s as d e s c r i b e d b y o t h e r a u t h o r s (5, 6, 8) w e r e o b s e r v e d in t h e p e r i o d
Our results show that inflammation of the ventricular system (especially the choroid plexus) and meninges, with the presence of intracellular
Table 1: Intensity of inflammatory lesions, immunoperoxidase reaction and bacterial counts in mice inoculated sub-
cutaneously with 1 x 10 9 cfu of Listeria monocytogenes. Day
Mouse no.
Ventricles PAP
HE
1
2 3
4 5
6
7
8
9
1 2 3 4 5 6 7 8 9 10 11 12 13" 14" 15" 16" 17" 18 19 20* 21" 22 23* 24* 25* 26* 27
Meninges
Parenchyma
Liver
Spleen
--
. . . . . . . . . + + . + + + +++ +++ . ++ +++ +++ . +++ +++ +++ +++ .
. . . . . . . . .
HE . . .
. . . . . . + +
.
. + + + ++ ++
.
. + +++ +++
.
. +++ +++ +++ +++
.
.
. . .
PAP . . .
HE . . .
. . . . . . . . . . . . . . . . . . + + . . . . . . . + + . . . . + + + + + + . . . + + ++ ++ ++ ++ . . . ++ ++ ++ ++ + + + + . . .
PAP . . .
PAP
. . .
+
+
-
-
-
-
+ -
+ -
+
+ .
HE
+/+ + + + + ++ + + + + ++ ++ ++ ++ ++ ++ + + ++ + + + .
+ + +/+/+ +/+ + + ++ ++ ++ + + + + ++ + +/-
Log cfutg 3.70 3.66 3.61 5.20 5.65 5.42 5.81 5.69 5.84 6.11 5.91 5.83 7.43 ND 7.26 6.01 5.65 5.70 5.12 ND 5.21 3.30 2.94 ND ND 2.81 2.89
HE + + ++ ++ ++ ++ +++ ++ +++ +++ +++ +++ +++ ++ ++ + +++ +++ +++ ++ + + -
PAP + + + + + + + + + +++ ++ ++ + + + +/+ + + + -
Log efulg 4.95 4.78 5.01 7.21 6.96 7.18 7.70 7.90 7.58 8.20 7.95 7.74 8.83 ND 9.21 7.20 6.60 6.27 6.05 ND 5.93 3.34 3.03 ND ND 2.24 2.46
HE: hematoxylin-eosin; PAP: peroxidase-antiperoxidase -: no lesion (HE)/no reaction (PAP); +/-: minimal lesion/reaction; +: low grade lesion/reaction; ++: moderate lesion/reaction; +++: intense lesion/reaction; ND: not done (autolytie); *: died.
746
Eur. J. Clin. Microbiol. Infect. Dis.
A
B
Figure 1: A: Third ventricle of a Listeria monocytogenes-infected mouse (day 6 post-inoculation) showing mild choroiditis. Note inflammatory cells in choroid plexus and in lumen. B: Lateral ventricle of a Listeria monocytogenes-infeeted mouse (day 9 post-inoculation). A profuse exudation of inflammatory cells into the ventricular space and necrosis of the choroidal epithelial cells (arrow) can be seen (Bar = 50 •m).
Listeria monocytogenes within phagocytic cells, is the most relevant pathological finding in the CNS after subcutaneous inoculation in mice. Encephalitis in this murine model was not as frequent and intense as described in the literature (3). Inflammatory lesions of the ports and medulla oblongata, as described in the rhombencephalitic form of small-ruminant listeriosis, were not observed. The incidence and severity of neurological symptoms and lesions increased when the bacterial counts in the liver and spleen began to decline, showing that CNS involvement is a late
phenomenon in septicemic listeriosis. Using the subcutaneous route, the course of the infection, as determined from peak multiplication of Listeria monocytogenes in the liver and spleen, is slightly retarded compared to infection after intravenous or intraperitoneal inoculation (4, 5, 7). This fact might explain the high rate of CNS symptoms and lesions seen in our experiments. The choroid plexus plays an essential role in the development of meningitis in other experimental models of bacterial infection (9, 10). Choroiditis is the first and most constant lesion produced by
Vol. 11, 1992
Streptococcus suis type 2 in CNS (10), and intracellular Streptococcus suis type 2 organisms are always associated with the lesions. T h e s e investigations indicated that the choroid plexus is the site at which Streptococcus suis type 2 within m o n o n u c l e a r phagocytes gains access to cerebrospinal fluid (CSF) spaces, O u r data suggest that Listeria rnonocytogenes also reaches the choroid plexus within m o n o n u c l e a r phagocytes, probably along the normal migration routes o f these ceils (11), causing choroiditis, ependymitis, meningitis and finally encephalitis. T h e intensity of the inflammatory lesions in the choroid plexus is presumably greater than in other ependymal structures whereas meningitis is of moderate severity, and encephalitis is infrequent and much less severe. O t h e r data support these assumptions: 1) the presence of free Listeria monocytogenes organisms in the bloodstream is known to be only a transient phase in experimental listeriosis (4, 5); 2) most organisms (90 % ) are captured and killed after intravenous inoculation by macrophages resident in the liver and spleen (5, 8); 3) surviving Listeria monocytogenes begin a rapid multiplication within the resident or inflammatory macrophages (5, 12); 4) Listeria monocytogenes-laden m o n o n u c l e a r phagocytes transport bacteria throughout the host organism, especially to sites, such as lymphoid organs (13), in which the presence of mononuclear phagocytes is a normal feature. In the CNS, a population of m o n o n u c l e a r phagocytes can be found associated with the epithelium of the ventricular system, particularly, the epithelium of the choroid plexus (11). The presence of such cells underlying the ependymal epithelium strongly suggests that they may play a role in CNS involvement during systemic listeriosis. In conclusion, our findings are in agreement with the hypothesis that mononuclear phagocytes and the choroid plexus play a substantial role in the pathogenic mechanism of some bacterial meningitides (10). Furthermore, choroiditis and meningitis can be r e p r o d u c e d by subcutaneous inoculation in the Swiss CD1 strain of mice, this being in
747
our opinion a suitable model for listeric meningitis in humans and other species.
References
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