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Cerebral Listeriosis in an Adult Freiberger Gelding
J. Comp. Path. 2006, Vol. 134, 249–253
SHORT PAPER
www.elsevier.com/locate/jcpa
Cerebral Listeriosis in an Adult Freiberger Gelding M. Ru¨tten, A. Lehner*, A. Pospischil and T. Sydler Institutes of Veterinary Pathology and *Institute of Food Safety and Hygiene, Vetsuisse Faculty, University of Zu¨rich, Winterthurerstrasse 268, CH-8057 Zu¨rich, Switzerland
Summary Listeria monocytogenes infection, although rare in horses, can lead to septicaemia, gastroenteritis and abortion or stillborn foals. Equine cerebral listeriosis has been reported, but only in newborn animals. This report describes a Freiberger gelding with severe neuronal symptoms of sudden onset. The animal collapsed within 24 h and was humanely killed. Necropsy revealed multiple small brown to reddish foci within the brain stem and pons. Histopathology demonstrated multifocal suppurative meningoencephalitis with microabscesses and occasional intra-lesional, coccoid to rod-shaped, bacteria. These were identified immunohistochemically as Listeria spp. and further specified as L. monocytogenes by a commercial test system based on in-situ hybridization. q 2005 Elsevier Ltd. All rights reserved. Keywords: bacterial infection; cerebral listeriosis; horse; Listeria monocytogenes
Introduction Listeria monocytogenes is a ubiquitous, gram-positive, facultative intracellular bacterium with a worldwide distribution, and has been isolated from diseased mammals and birds of many species. Equine listeriosis is rarely reported, but its manifestations are similar to those in ruminants and other herbivores (Emerson and Jarvis, 1968; Mayer et al. 1975, 1976). Reports of L. monocytogenes infections in horses include septicaemia in newborn foals (Wallace and Hathcock, 1995; Wilkins et al., 2000; Jose-Cunilleras and Hinchcliff, 2001), and septicaemia and gastroenteritis in older animals (Gudmundsdottir et al., 2004). There are a few reports of abortions and weak neonatal foals due to intrauterine infections with L. monocytogenes (Mayer et al., 1976; Welsh, 1983), but, as in ruminants, intrauterine infections do not seem to reduce fertility (Mason et al., 1980). The organism was also isolated from a case of keratitis in a horse (Sanchez et al., 2001). Neurological disease caused by L. monocytogenes has been described in newborn foals (Wallace and Hathcock, 1995; Jose-Cunilleras and Hinchcliff, 2001) and in an immunodeficient 0021-9975/$ - see front matter
doi:10.1016/j.jcpa.2005.09.007
Arabian foal aged one month (Clark et al., 1978), but not in older animals. To our knowledge, the description that follows is the first of cerebral listeriosis in an adult horse. The animal, a 6-year-old Freiberger gelding, suddenly became severely lame in the left hind leg and was found the next day leaning against the wall of its box, its head turned to the right, nostrils twisted to the same side, and mydriasis in the right eye. It then collapsed and was humanely killed.
Materials and Methods Necropsy, Specimens and Histopathology Post-mortem specimens consisted of heart, liver, spleen, lung, kidney, spinal cord, brain and the ganglion trigeminale. They were fixed in 4% buffered formalin for 24 h (72 h for central nervous system [CNS]), embedded in paraffin wax and processed by routine methods. Sections (2–3 mm) were stained with haematoxylin and eosin (HE) for histopathology. In addition, sections of medulla oblongata and pons were stained by the silver method of Steiner (Weilenmann, 1989). q 2005 Elsevier Ltd. All rights reserved.
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M. Ru¨tten et al.
Immunohistochemistry (IHC) CNS specimens with histopathological lesions (see below) suggestive of listeriosis were processed for immunoperoxidase labelling as previous described (Ehrensperger et al., 2001). Briefly, sections (2– 3 mm) were mounted at 378C on positively charged glass slides (Superfrost Color; Menzel, Braunschweig, Germany), dried and lightly stained with haematoxylin. They were treated with Pronase (protease Type XXVIII; Sigma, Buchs, Switzerland) 0.1% for 5 min to expose antigenic structures, and endogenous peroxidase was blocked with H2O2 3% in a 0.2% aqueous solution of sodium azide for 10 min at room temperature. A polyclonal rabbit antiserum against Listeria spp. (Domingo et al., 1986) was kindly supplied by Dr M. Domingo. The slides were incubated for 1 h with the primary antibody, diluted 1 in 500. The secondary antibody was a biotinylated anti-mouse/rabbit mixture (Dako ChemMate TM; Dako Diagnostics, Zug, Switzerland) with which the slides were incubated for 15 min at room temperature, followed by treatment with streptavidin-peroxidase for 15 min and, finally, with the peroxidase substrate aminoethyl-carbazol (AEC [red] Substrate Kit; Zymed Laboratories, Basel, Switzerland, for 15 min. The slides were washed with phosphate-buffered saline (PBS), pH 8.0 between each step. Fluorescence In-situ Hybridization (FISH) CNS sections (2–3 mm) were mounted on positively charged glass slides (Superfrost Color; Menzel) at 60 8C. The sections were dewaxed with xylene. The VITw Listeria monocytogenes kit (Vermicon, Munich, Germany) was employed for in-situ analysis
Fig. 1. Brain stem, showing multiple microabscesses within the parenchyma, and perivascular cuffing. HE. Bar, 100 mm.
according to the manufacturer’s instructions with small modifications (Stephan et al., 2003). The volumes of solution B2 and solution VITLmo were ca 100 ml/section, so that each tissue section was completely covered. The method is based on the principle that gene probes specifically designed for both the genus Listeria, and the species L. monocytogenes, penetrate the bacteria and bind to the ribosomal RNA at the target points within the bacteria. During subsequent analysis with fluorescence confocal microscopy (Leica TCS SP2 AOBS; Leica Microsystems, Mannheim, Germany), Listeria spp. labelled with the genus-specific probe, when excited with the 488 nm laser line, emit a green fluorescence (emission 500–540 nm); and L. monocytogenes labelled with the species-specific probe, when excited with the 543 nm laser line, emit a red fluorescence (emission 550–630 nm). Image stacks (3D) were collected sequentially (to prevent green–red channel spill-over) and prepared with Imaris Easy-3D (Bitplane, Zu¨rich, Switzerland). With this procedure, in-focus images of all positively labelled bacteria present in a 2– 3 mm thick section could be combined in a single 2D image, facilitating analysis.
Results Gross Findings After slicing the brain stem, numerous, small (%1 mm), brown to red foci were visible. There were no other significant gross lesions. Histological Findings The lesions were restricted to the CNS. In the brain stem, and in particular the medulla and pons, multiple, randomly distributed, small to coalescing foci of liquefaction necrosis infiltrated by large numbers of neutrophils (so-called microabscesses), were present. In the adjacent parenchyma, multifocal disseminated gliosis, diffuse infiltrations of neutrophils, and macrophages were evident. There was marked degeneration of neurons close to the microabscesses. In the suppurative foci, the vessel walls were enlarged by hypertrophy of the endothelial cells. Many vessels were surrounded by perivascular cuffs, which consisted mainly of lymphocytes, plasma cells, neutrophils and histiocytes (Fig. 1). The meninges were oedematous and showed infiltrates of inflammatory cells such as neutrophils, lymphocytes, plasma cells and macrophages. The blood vessels were slightly congested. The Steiner staining method revealed a few intraand extra-cellular, coccoid to rod-shaped bacteria
251
Equine Cerebral Listeriosis
FISH Probes specific for the genus Listeria spp. (green fluorescence) and the species L. monocytogenes (red) were co-localized to spots (ca 1 mm), distributed singly or in clusters around nuclei (blue) within the same regions of the medulla oblongata and pons as those examined by IHC (Fig. 4a and b). These results provided clear evidence of L. monocytogenes in the lesions.
Discussion
Fig. 2. Brain stem, showing a few intra- and extra-cellular coccoid to rod-shaped bacteria (arrowheads), Steiner staining. Bar, 10 mm.
within the lesions (Fig. 2). In the lung, a slight perivascular infiltration of lymphocytes and plasma cells was seen, as was eosinophilic, homogeneous fluid in the alveolar lumina. The oedema in the lung and congestion in the spleen were interpreted as side effects of euthanasia. The other organs showed no lesions.
In cattle and small ruminants, encephalitic listeriosis is sporadic but well known. In contrast, in horses disease caused by L. monocytogenes is rare. However, septicaemia in adult horses and foals associated with diarrhoea, fever, mild colic, restlessness, laboured ventilation and jaundice has been described (Emerson and Jarvis, 1968; Wallace
IHC The L. monocytogenes antibody revealed small intraand extra-cellular, pleomorphic, round-shaped bacteria within microabscesses in the medulla oblongata and pons (Fig. 3). The distribution of bacteria corresponded generally with that demonstrated by Steiner staining.
Fig. 3. Brain stem, showing pleomorphic plump rods (Listeria spp.). IHC. Bar, 10 mm.
Fig. 4. Brain stem, showing pleomorphic rods (a) fluorescing green (for the genus Listeria), nuclei fluorescing blue, and (b) fluorescing red (for the species L. monocytogenes), with nuclei fluorescing blue. FISH. Bar, 20 mm.
252
M. Ru¨tten et al.
and Hathcock, 1995; Jose-Cunilleras and Hinchcliff, 2001; Gudmundsdottir et al., 2004). In ruminants, the characteristic signs of cerebral listeriosis are leaning against a post or fence and wandering in circles (“circling disease”). An affected animal often holds its head to one side or rests it on the floor or against the wall (“pushing syndrome”). Subsequently, ataxia, incoordination, cranial nerve deficiencies, tremor and states of excitation may occur, followed by coma and death (Rosenberger, 1994). The Freiberger gelding described here showed the characteristic signs as described for cattle and small ruminants with cerebral listeriosis. The distribution and nature of the inflammation were also comparable with those described in cattle and small ruminants (Johnson et al., 1996; Ehrensperger et al., 2001; Campero et al., 2002). Ehrensperger et al. (2001) reported that the severity of the lesions in sheep and goats with cerebral listeriosis was related to the number of intra-lesional bacteria. They distinguished between a form consisting almost exclusively of microabscesses, a form consisting only of multifocal microgliosis or an intermediate form with features of both. High numbers of bacteria were particularly apparent in microabscesses. The horse described here showed an acute fulminant suppurative inflammation, which took the form of microabscesses, but the numbers of visible intra-lesional L. monocytogenes were low by comparison with those described by Ehrensperger et al. (2001). The gelding was reported to have received hay silage, consisting of the small bales that are commonly fed to ruminants, especially cattle, in Switzerland. L. monocytogenes is commonly found in normal animals in the tonsils and gut-associated lymphoid tissue, and in large numbers in faeces. Infection typically occurs by ingestion, often as the result of faecal contamination. In central Europe, the disease is most common in winter or early spring. The bacteria invade the apical side of intestinal epithelial cell (Daniels et al., 2000) and penetrate the intestinal wall. Dissemination to the neighbouring cells occurs via plasma membrane protrusions. In this way, the organisms avoid the host defence mechanisms. Damaged oral, nasal or ocular mucosa is also suspected as a possible entry route, especially in cases of CNS infection. The organisms then gain access to the CNS via branches of the trigeminal nerve. Feeding of silage is well known to be a risk factor for listeriosis, particularly when the silage is of poor quality (pH above 5.5) (Ryser et al., 1997). It would seem to have been the most likely source of infection in the present case.
The reason why listeriosis is rarely seen in horses may be related to the feeding management. In Switzerland, horses are infrequently fed with silage and almost never with corn silage, which is heavily and frequently contaminated with L. monocytogenes (Ryser et al., 1997; Boerlin et al., 2003). In addition, horses often differ from ruminants in being kept under more extensive and more hygienic conditions. Nevertheless, equine listeriosis was recognized in Iceland, where large herds of horses are fed grass silage during the winter (Gudmundsdottir et al., 2004); unfortunately no histological findings were given in this report of 19 horses in which Listeria spp. were isolated from the inner organs and faeces.
Acknowledgments We thank Monika Hilbe and Kati Zlinszky for processing the specimens for immunohistochemistry. We also thank Lloyd Vaughan for the confocal images, for reading the manuscript, and for useful suggestions.
References Boerlin, P., Boerlin-Petzhold, F. and Jemmi, T. (2003). Use of listeriolysin O and internalin A in a seroepidemiological study of listeriosis in Swiss dairy cows. Journal of Clinical Microbiology, 41, 1055– 1061. Campero, C. M., Odeo´n, A. C., Cipolla, A. L., Moore, D. P., Poso, M. A. and Odriozola, E. (2002). Demonstration of Listeria monocytogenes by immunohistochemistry in formalin-fixed brain tissues from natural cases of ovine and bovine encephalitis. Journal of Veterinary Medicine (B), 49, 379–383. Clark, E. G., Turner, A. S., Boyson, B. G. and Rouse, B. T. (1978). Listeriosis in an Arabian foal with combined immunodeficiency. Journal of the American Veterinary Medical Association, 172, 363–366. Daniels, J. J. D., Autenrieth, I. B. and Goebel, W. (2000). Interaction of Listeria monocytogenes with the intestinal epithelium. FEMS Microbiology Letters, 190, 323–328. Domingo, M., Ramos, J. A., Dominguez, L., Ferrer, L. and Marco, A. (1986). Demonstration of Listeria monocytogenes with the PAP technique in formalin fixed and paraffin embedded tissues of experimentally infected mice. Journal of Veterinary Medicine (B), 33, 537–542. Ehrensperger, F., Hilbe, M., Sydler, T., Corboz, L., Braun, U. and Pospischil, A. (2001). Zerebrale Listeriose bei Schaf und Ziege: eine histopathologische und immunhistologische Studie. Wiener Tiera¨rztliche Monatsschrift, 88, 219–225.
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Emerson, F. G. and Jarvis, A. (1968). Listeriosis in ponies. Journal of the American Veterinary Medical Association, 152, 1645–1646. Gudmundsdottir, K. B., Svansson, V., Aalbæk, B., Gunnarsson, E. and Sigurdarson, S. (2004). Listeria monocytogenes in horses in Iceland. Veterinary Record, 155, 456–459. Johnson, G. C., Maddox, C. W., Fales, W. H., Wolff, W. A., Randle, R. F., Ramos, J. A., Schwartz, H., Heise, K. M., Beatz, A. L., Wesley, I. V. and Wagner, D. E. (1996). Epidemiologic evaluation of encephalitic listeriosis in goats. Journal of the American Veterinary Medical Association, 208, 1695–1699. Jose-Cunilleras, E. and Hinchcliff, K. W. (2001). Listeria monocytogenes septicaemia in foals. Equine Veterinary Journal, 33, 519–522. Mason, R. W., Brennan, R. G. and Cordould, A. (1980). Listeria monocytogenes abortion in a mare. Australian Veterinary Journal, 56, 613. Mayer, H., Kinzler, M. and Sickel, E. (1976). Listeriose in einem Reitpferdebestand. Berliner und Mu¨nchner Tiera¨rztlicher Wochenschrift, 89, 209–211. Mayer, H., Seelinger, H. P. R., Sickel, E. and Kinzler, M. (1975). Serologische Untersuchungen u¨ber Listeriose in Pferdebesta¨nden. Berliner und Mu¨nchner Tiera¨rztlicher Wochenschrift, 88, 345–347. Rosenberger, G. (1994). Listeriose. In: Krankheiten des Rindes, 3rd Edit. G. Rosenberger, G. Dirkson, H. D. Gru¨nder and M. Sto¨ber, Eds, Blackwell Verlag, Berlin, pp. 826–832.
Ryser, E. T., Arimi, S. M. and Donnelly, C. W. (1997). Effects of pH on distribution of listeria ribotypes in corn, hay and grass silage. Applied and Environmental Microbiology, 63, 3695–3697. Sanchez, S., Studer, M., Barlett, P. and Bounous, D. (2001). Listeria keratitis in a horse. Veterinary Ophthalmology, 4, 217–219. Stephan, R., Schumacher, S. and Zychowska, A. (2003). The VITw technology for rapid detection of Listeria monocytogenes and other Listeria spp. International Journal of Food Microbiology, 89, 287–290. Wallace, S. S. and Hathcock, T. L. (1995). Listeria monocytogenes septicemia in a foal. Journal of the American Veterinary Medical Association, 207, 1325–1326. Weilenmann, R. (1989). Histotechnik. In: Laboratoriumsmedizin, 3rd Edit. P. Hagemann and K. Rosenmund, Eds, Hirzel, Stuttgart, p. 62. Welsh, R. D. (1983). Equine abortion caused by Listeria monocytogenes serotype 4. Journal of the American Veterinary Medical Association, 182, 291. Wilkins, P. A., Marsh, P. S., Acland, H. and Del Piero, F. (2000). Listeria monocytogenes septicemia in a Thoroughbred foal. Journal of Veterinary Diagnostic Investigation, 12, 173–176.
Received; May 19th; 2005 Accepted; September 27th; 2005
SHORT PAPER
www.elsevier.com/locate/jcpa
Cerebral Listeriosis in an Adult Freiberger Gelding M. Ru¨tten, A. Lehner*, A. Pospischil and T. Sydler Institutes of Veterinary Pathology and *Institute of Food Safety and Hygiene, Vetsuisse Faculty, University of Zu¨rich, Winterthurerstrasse 268, CH-8057 Zu¨rich, Switzerland
Summary Listeria monocytogenes infection, although rare in horses, can lead to septicaemia, gastroenteritis and abortion or stillborn foals. Equine cerebral listeriosis has been reported, but only in newborn animals. This report describes a Freiberger gelding with severe neuronal symptoms of sudden onset. The animal collapsed within 24 h and was humanely killed. Necropsy revealed multiple small brown to reddish foci within the brain stem and pons. Histopathology demonstrated multifocal suppurative meningoencephalitis with microabscesses and occasional intra-lesional, coccoid to rod-shaped, bacteria. These were identified immunohistochemically as Listeria spp. and further specified as L. monocytogenes by a commercial test system based on in-situ hybridization. q 2005 Elsevier Ltd. All rights reserved. Keywords: bacterial infection; cerebral listeriosis; horse; Listeria monocytogenes
Introduction Listeria monocytogenes is a ubiquitous, gram-positive, facultative intracellular bacterium with a worldwide distribution, and has been isolated from diseased mammals and birds of many species. Equine listeriosis is rarely reported, but its manifestations are similar to those in ruminants and other herbivores (Emerson and Jarvis, 1968; Mayer et al. 1975, 1976). Reports of L. monocytogenes infections in horses include septicaemia in newborn foals (Wallace and Hathcock, 1995; Wilkins et al., 2000; Jose-Cunilleras and Hinchcliff, 2001), and septicaemia and gastroenteritis in older animals (Gudmundsdottir et al., 2004). There are a few reports of abortions and weak neonatal foals due to intrauterine infections with L. monocytogenes (Mayer et al., 1976; Welsh, 1983), but, as in ruminants, intrauterine infections do not seem to reduce fertility (Mason et al., 1980). The organism was also isolated from a case of keratitis in a horse (Sanchez et al., 2001). Neurological disease caused by L. monocytogenes has been described in newborn foals (Wallace and Hathcock, 1995; Jose-Cunilleras and Hinchcliff, 2001) and in an immunodeficient 0021-9975/$ - see front matter
doi:10.1016/j.jcpa.2005.09.007
Arabian foal aged one month (Clark et al., 1978), but not in older animals. To our knowledge, the description that follows is the first of cerebral listeriosis in an adult horse. The animal, a 6-year-old Freiberger gelding, suddenly became severely lame in the left hind leg and was found the next day leaning against the wall of its box, its head turned to the right, nostrils twisted to the same side, and mydriasis in the right eye. It then collapsed and was humanely killed.
Materials and Methods Necropsy, Specimens and Histopathology Post-mortem specimens consisted of heart, liver, spleen, lung, kidney, spinal cord, brain and the ganglion trigeminale. They were fixed in 4% buffered formalin for 24 h (72 h for central nervous system [CNS]), embedded in paraffin wax and processed by routine methods. Sections (2–3 mm) were stained with haematoxylin and eosin (HE) for histopathology. In addition, sections of medulla oblongata and pons were stained by the silver method of Steiner (Weilenmann, 1989). q 2005 Elsevier Ltd. All rights reserved.
250
M. Ru¨tten et al.
Immunohistochemistry (IHC) CNS specimens with histopathological lesions (see below) suggestive of listeriosis were processed for immunoperoxidase labelling as previous described (Ehrensperger et al., 2001). Briefly, sections (2– 3 mm) were mounted at 378C on positively charged glass slides (Superfrost Color; Menzel, Braunschweig, Germany), dried and lightly stained with haematoxylin. They were treated with Pronase (protease Type XXVIII; Sigma, Buchs, Switzerland) 0.1% for 5 min to expose antigenic structures, and endogenous peroxidase was blocked with H2O2 3% in a 0.2% aqueous solution of sodium azide for 10 min at room temperature. A polyclonal rabbit antiserum against Listeria spp. (Domingo et al., 1986) was kindly supplied by Dr M. Domingo. The slides were incubated for 1 h with the primary antibody, diluted 1 in 500. The secondary antibody was a biotinylated anti-mouse/rabbit mixture (Dako ChemMate TM; Dako Diagnostics, Zug, Switzerland) with which the slides were incubated for 15 min at room temperature, followed by treatment with streptavidin-peroxidase for 15 min and, finally, with the peroxidase substrate aminoethyl-carbazol (AEC [red] Substrate Kit; Zymed Laboratories, Basel, Switzerland, for 15 min. The slides were washed with phosphate-buffered saline (PBS), pH 8.0 between each step. Fluorescence In-situ Hybridization (FISH) CNS sections (2–3 mm) were mounted on positively charged glass slides (Superfrost Color; Menzel) at 60 8C. The sections were dewaxed with xylene. The VITw Listeria monocytogenes kit (Vermicon, Munich, Germany) was employed for in-situ analysis
Fig. 1. Brain stem, showing multiple microabscesses within the parenchyma, and perivascular cuffing. HE. Bar, 100 mm.
according to the manufacturer’s instructions with small modifications (Stephan et al., 2003). The volumes of solution B2 and solution VITLmo were ca 100 ml/section, so that each tissue section was completely covered. The method is based on the principle that gene probes specifically designed for both the genus Listeria, and the species L. monocytogenes, penetrate the bacteria and bind to the ribosomal RNA at the target points within the bacteria. During subsequent analysis with fluorescence confocal microscopy (Leica TCS SP2 AOBS; Leica Microsystems, Mannheim, Germany), Listeria spp. labelled with the genus-specific probe, when excited with the 488 nm laser line, emit a green fluorescence (emission 500–540 nm); and L. monocytogenes labelled with the species-specific probe, when excited with the 543 nm laser line, emit a red fluorescence (emission 550–630 nm). Image stacks (3D) were collected sequentially (to prevent green–red channel spill-over) and prepared with Imaris Easy-3D (Bitplane, Zu¨rich, Switzerland). With this procedure, in-focus images of all positively labelled bacteria present in a 2– 3 mm thick section could be combined in a single 2D image, facilitating analysis.
Results Gross Findings After slicing the brain stem, numerous, small (%1 mm), brown to red foci were visible. There were no other significant gross lesions. Histological Findings The lesions were restricted to the CNS. In the brain stem, and in particular the medulla and pons, multiple, randomly distributed, small to coalescing foci of liquefaction necrosis infiltrated by large numbers of neutrophils (so-called microabscesses), were present. In the adjacent parenchyma, multifocal disseminated gliosis, diffuse infiltrations of neutrophils, and macrophages were evident. There was marked degeneration of neurons close to the microabscesses. In the suppurative foci, the vessel walls were enlarged by hypertrophy of the endothelial cells. Many vessels were surrounded by perivascular cuffs, which consisted mainly of lymphocytes, plasma cells, neutrophils and histiocytes (Fig. 1). The meninges were oedematous and showed infiltrates of inflammatory cells such as neutrophils, lymphocytes, plasma cells and macrophages. The blood vessels were slightly congested. The Steiner staining method revealed a few intraand extra-cellular, coccoid to rod-shaped bacteria
251
Equine Cerebral Listeriosis
FISH Probes specific for the genus Listeria spp. (green fluorescence) and the species L. monocytogenes (red) were co-localized to spots (ca 1 mm), distributed singly or in clusters around nuclei (blue) within the same regions of the medulla oblongata and pons as those examined by IHC (Fig. 4a and b). These results provided clear evidence of L. monocytogenes in the lesions.
Discussion
Fig. 2. Brain stem, showing a few intra- and extra-cellular coccoid to rod-shaped bacteria (arrowheads), Steiner staining. Bar, 10 mm.
within the lesions (Fig. 2). In the lung, a slight perivascular infiltration of lymphocytes and plasma cells was seen, as was eosinophilic, homogeneous fluid in the alveolar lumina. The oedema in the lung and congestion in the spleen were interpreted as side effects of euthanasia. The other organs showed no lesions.
In cattle and small ruminants, encephalitic listeriosis is sporadic but well known. In contrast, in horses disease caused by L. monocytogenes is rare. However, septicaemia in adult horses and foals associated with diarrhoea, fever, mild colic, restlessness, laboured ventilation and jaundice has been described (Emerson and Jarvis, 1968; Wallace
IHC The L. monocytogenes antibody revealed small intraand extra-cellular, pleomorphic, round-shaped bacteria within microabscesses in the medulla oblongata and pons (Fig. 3). The distribution of bacteria corresponded generally with that demonstrated by Steiner staining.
Fig. 3. Brain stem, showing pleomorphic plump rods (Listeria spp.). IHC. Bar, 10 mm.
Fig. 4. Brain stem, showing pleomorphic rods (a) fluorescing green (for the genus Listeria), nuclei fluorescing blue, and (b) fluorescing red (for the species L. monocytogenes), with nuclei fluorescing blue. FISH. Bar, 20 mm.
252
M. Ru¨tten et al.
and Hathcock, 1995; Jose-Cunilleras and Hinchcliff, 2001; Gudmundsdottir et al., 2004). In ruminants, the characteristic signs of cerebral listeriosis are leaning against a post or fence and wandering in circles (“circling disease”). An affected animal often holds its head to one side or rests it on the floor or against the wall (“pushing syndrome”). Subsequently, ataxia, incoordination, cranial nerve deficiencies, tremor and states of excitation may occur, followed by coma and death (Rosenberger, 1994). The Freiberger gelding described here showed the characteristic signs as described for cattle and small ruminants with cerebral listeriosis. The distribution and nature of the inflammation were also comparable with those described in cattle and small ruminants (Johnson et al., 1996; Ehrensperger et al., 2001; Campero et al., 2002). Ehrensperger et al. (2001) reported that the severity of the lesions in sheep and goats with cerebral listeriosis was related to the number of intra-lesional bacteria. They distinguished between a form consisting almost exclusively of microabscesses, a form consisting only of multifocal microgliosis or an intermediate form with features of both. High numbers of bacteria were particularly apparent in microabscesses. The horse described here showed an acute fulminant suppurative inflammation, which took the form of microabscesses, but the numbers of visible intra-lesional L. monocytogenes were low by comparison with those described by Ehrensperger et al. (2001). The gelding was reported to have received hay silage, consisting of the small bales that are commonly fed to ruminants, especially cattle, in Switzerland. L. monocytogenes is commonly found in normal animals in the tonsils and gut-associated lymphoid tissue, and in large numbers in faeces. Infection typically occurs by ingestion, often as the result of faecal contamination. In central Europe, the disease is most common in winter or early spring. The bacteria invade the apical side of intestinal epithelial cell (Daniels et al., 2000) and penetrate the intestinal wall. Dissemination to the neighbouring cells occurs via plasma membrane protrusions. In this way, the organisms avoid the host defence mechanisms. Damaged oral, nasal or ocular mucosa is also suspected as a possible entry route, especially in cases of CNS infection. The organisms then gain access to the CNS via branches of the trigeminal nerve. Feeding of silage is well known to be a risk factor for listeriosis, particularly when the silage is of poor quality (pH above 5.5) (Ryser et al., 1997). It would seem to have been the most likely source of infection in the present case.
The reason why listeriosis is rarely seen in horses may be related to the feeding management. In Switzerland, horses are infrequently fed with silage and almost never with corn silage, which is heavily and frequently contaminated with L. monocytogenes (Ryser et al., 1997; Boerlin et al., 2003). In addition, horses often differ from ruminants in being kept under more extensive and more hygienic conditions. Nevertheless, equine listeriosis was recognized in Iceland, where large herds of horses are fed grass silage during the winter (Gudmundsdottir et al., 2004); unfortunately no histological findings were given in this report of 19 horses in which Listeria spp. were isolated from the inner organs and faeces.
Acknowledgments We thank Monika Hilbe and Kati Zlinszky for processing the specimens for immunohistochemistry. We also thank Lloyd Vaughan for the confocal images, for reading the manuscript, and for useful suggestions.
References Boerlin, P., Boerlin-Petzhold, F. and Jemmi, T. (2003). Use of listeriolysin O and internalin A in a seroepidemiological study of listeriosis in Swiss dairy cows. Journal of Clinical Microbiology, 41, 1055– 1061. Campero, C. M., Odeo´n, A. C., Cipolla, A. L., Moore, D. P., Poso, M. A. and Odriozola, E. (2002). Demonstration of Listeria monocytogenes by immunohistochemistry in formalin-fixed brain tissues from natural cases of ovine and bovine encephalitis. Journal of Veterinary Medicine (B), 49, 379–383. Clark, E. G., Turner, A. S., Boyson, B. G. and Rouse, B. T. (1978). Listeriosis in an Arabian foal with combined immunodeficiency. Journal of the American Veterinary Medical Association, 172, 363–366. Daniels, J. J. D., Autenrieth, I. B. and Goebel, W. (2000). Interaction of Listeria monocytogenes with the intestinal epithelium. FEMS Microbiology Letters, 190, 323–328. Domingo, M., Ramos, J. A., Dominguez, L., Ferrer, L. and Marco, A. (1986). Demonstration of Listeria monocytogenes with the PAP technique in formalin fixed and paraffin embedded tissues of experimentally infected mice. Journal of Veterinary Medicine (B), 33, 537–542. Ehrensperger, F., Hilbe, M., Sydler, T., Corboz, L., Braun, U. and Pospischil, A. (2001). Zerebrale Listeriose bei Schaf und Ziege: eine histopathologische und immunhistologische Studie. Wiener Tiera¨rztliche Monatsschrift, 88, 219–225.
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Received; May 19th; 2005 Accepted; September 27th; 2005