key: cord-285493-eg2ltip6
authors: Schwab, S.; Herden, C.; Seeliger, F.; Papaioannou, N.; Psalla, D.; Polizopulou, Z.; Baumgärtner, W.
title: Non-suppurative Meningoencephalitis of Unknown Origin in Cats and Dogs: an Immunohistochemical Study
date: 2007-02-01
journal: J Comp Pathol
DOI: 10.1016/j.jcpa.2006.11.006
sha: 
doc_id: 285493
cord_uid: eg2ltip6

Non-suppurative meningoencephalitis of unknown cause is a frequent finding in dogs and cats. Fifty-three dogs and 33 cats with non-suppurative meningoencephalitis of unknown aetiology were examined immunohistochemically for 18 different infectious agents, including viruses, bacteria and prion protein(Sc). In 14 (26%) of the dogs and 13 (39%) of the cats a causative agent was identified in the central nervous system (CNS), two dogs and one cat giving positive results for two infectious agents simultaneously. The study revealed infections with known causative agents (porcine herpes virus 1, feline infectious peritonitis virus, Escherichia coli) and a new disease pattern of parvovirus infection in the CNS of dogs and cats. Infection of the CNS with feline leukaemia virus was found in a cat. Five dogs and four cats gave positive results for West Nile virus (WNV) antigen. In one dog, canine parainfluenza virus antigen was detected in the brain. Four dogs and four cats gave positive results for encephalomyocarditis virus (EMCV). The significance of the detection of WNV and EMCV antigen requires further study. The aetiology remained undetermined in 39 dogs (74%) and 20 cats (61%). Although it is possible that non-infectious causes play a more important role than previously thought, infections with hitherto unrecognized agents cannot be ruled out.

Non-suppurative meningoencephalitis in cats and dogs is usually considered to be caused by viral infections (Summers et al., 1995) , and the histopathological ¢ndings consist mainly of perivascular and parenchymal in¢ltration with lymphocytes, macrophages and plasma cells, usually associated with meningitis and occasionally with in£ammation of the plexus chorioideus and the ependyma (Braund, 1980; Luttgen, 1988) . Viruses, due to cell tropism and route of infection, often induce a typical pattern of in£ammation in the brain and spinal cord (Braund, 2001) .

In newborn animals, infections of the central nervous system (CNS) with bacteria, including various strains of Escherichia coli, are a common sequel to septicaemia, whereas in adult dogs and cats bacteria may follow a neurogenic, otogenic or rhinogenic route to enter the CNS (Meric,1988; Summers et al.,1995) . Whilst most bacterial CNS infections induce a purulent in-£ammation, some bacteria, particularly Listeria spp., as well as protozoa, fungi and algae, cause a non-suppurative, mainly granulomatous in£ammation of the CNS (Rand et al., 1994; Summers et al., 1995; Tipold, 1995; Quesnel et al., 1997) . In the brains of beagles with granulomatous leptomeningitis, Maeda et al. www.elsevier.com/locate/jcpa (1993) demonstrated E. coli and considered it to be the causative agent. If parasites enter the CNS, they often induce a granulomatous in£ammation (Summers et al., 1995) . In dogs, the most common parasite to cause infection of the CNS is Toxoplasma gondii, followed by Neospora caninum and, albeit rarely, Cuterebra spp. In cats, Cuterebra spp. are the most common parasites to be found in the CNS, followed by T. gondii in rare cases (Braund, 2001) .

There would also seem to be non-infectious causes of non-suppurative meningoencephalitis. Thus, autoimmune and hereditary mechanisms are suspected to be the underlying cause of CNS disorders such as granulomatous meningoencephalitis (GME) in dogs (Harris et al., 1988; Kipar et al., 1998) , pug dog encephalitis and non-suppurative meningoencephalitis in greyhounds (Callanan et al., 2002) . So far no aetiological agent has been detected for feline ''staggering disease'' or so-called feline polioencephalomyelitis (Vandevelde and Braund, 1979; Nowotny and Weissenbo º ck, 1995; Braund, 2001) , although some suspect staggering disease to be due to Borna disease virus (BDV) infection (Lundgren et al.,1995) . Paraneoplastic disorders of the CNS, well known in man, have not been reported in cats and dogs (Braund et al.,1987) .

Recent studies on a limited range of infectious agents failed to identify the cause of non-suppurative meningoencephalitis in 47% and 23% of dogs and cats, respectively (Rand et al., 1994; Tipold, 1995; Quesnel et al., 1997; K.Melzer, personal communication) . To provide further information, the present study sought to determine the cause of 86 cases of non-suppurative meningoencephalitis in dogs and cats by the immunohistochemical examination of formalin-¢xed, para⁄n wax-embedded tissue. In addition to examination for known viral and bacterial pathogens and prion protein Sc ,West Nile virus (WNV), a recently emerging cause of disease, was included in the present study (Komar, 2000; Lichtensteiger et al., 2003; Austgen et al., 2004) , together with encephalomyocarditis virus (EMCV), an agent so far demonstrated only in the CNS of pigs and rodents (Nowotny, 1996; Sue et al., 2003) .

Fifty-three canine and 33 feline cases of non-suppurative meningoencephalitis of unknown origin, collected during the period 1998^2003 by the Department of Pathology of the University of Veterinary Medicine Hannover, Germany, were selected. The case selection was made in the light of a preliminary investigation which revealed that 274 of 1209 (23%) dogs and 146 of 741 (20%) cats examined in a 5-year-period (1998^2002) displayed changes in the CNS. Non-suppurative meningoencephalitis accounted for 22% and 49% of the CNS changes in dogs and cats, respectively. The cause of 49 canine and 32 feline cases remained undetermined after examination by routine diagnostic procedures. These 81 animals, together with four dogs and one cat with undetermined non-suppurative meningoencephalitis encountered in 2003, were included in this study.

Due to the retrospective nature of the present study, clinical data were restricted to a short statement from the records of the Department of Pathology in most cases (46 dogs and 30 cats). In seven dogs and three cats, no clinical history was available.

Archived CNS sections of each case, stained with haematoxylin and eosin (HE), were re-examined for in-£ammatory changes. From each animal, one section of the cerebral cortex, the hippocampus (missing in three dogs and four cats), the midbrain (missing in ¢ve dogs and ¢ve cats), the cerebellum (missing in two dogs and two cats), the brain stem (missing in 17 dogs and 13 cats) and the spinal cord (available in only seven dogs and two cats) were examined. In one canine section, a spinal cord ganglion was present. The distribution of the lesions (e.g., polio-, leuco-, and panencephalitis) was categorized according to Summers et al. (1995) . The type of in£ammation was de¢ned as follows: lymphohistiocytic, if in¢ltrates consisted mainly of lymphocytes and macrophages; granulomatous, if macrophages predominated; pyogranulomatous, if neutrophils and macrophages were the main cell types; and mixed, if macrophages, lymphocytes, plasma cells and neutrophils appeared in equal quantities.

The degree of histological changes were determined semiquantitatively. Perivascular in¢ltrates were described as: mild (1^15 perivascular cells per Â100 ¢eld, or 1^2 cell layers per vessel, or both); moderate (16^30 perivascular cells per Â100 ¢eld or 2^3 cell layers per vessel, or both); and severe (430 perivascular cells per  100 ¢eld or X3 cell layers per vessel, or both). Meningeal in¢ltrates were described as mild (one or no more than a few mild in¢ltrates), moderate (one moderate in¢ltrate or several mild in¢ltrates), or severe (several moderate in¢ltrates or con£uent in¢ltrates).

If suggested by histological results, slides were additionally stained with periodic acid^Schi¡ (16 dogs, six cats), Ziehl^Neelsen (11dogs, one cat), Grocott (10 dogs, four cats), luxol-fast blue (nine dogs, one cat), cresylecht-violet (two dogs, one cat), alcian-blue (one cat) or Congo-red (one cat), as described by Romeis (1994) .

For the detection of 18 infectious agents (Table 1) , the avidin^biotin-peroxidase complex (ABC) method was performed, as previously described (Hsu et al., 1981; Wu º nschmann et al.,1997) . Due to their known cross-reactivity for the detection of canine and feline herpesvirus, parvovirus and coronavirus antigen, the same antibodies were used for both species. Feline tissue was not examined for canine adenovirus 1and canine tissue was not examined for feline leukaemia virus (FeLV). IHC for the detection of FeLVantigen was additionally performed on lymphoid tissue; however, such tissue was available from only 20 of the 33 cats. In one dog that showed immunoreactivity for porcine herpesvirus 1, tissue from the gastrointestinal tract was also examined, and in one dog and two cats showing immunoreactivity for EMCV, myocardial tissue was examined.

Brie£y, specimens were dewaxed in graded alcohols and endogenous peroxidase was quenched with H 2 O 2 0.5% in ethanol.The pre-treatments applied to sections (Table 2 ) depended on the primary antibody and were performed as previously described (see below). The various pre-treatments were as follows: Pronase E (Merck, Darmstadt, Germany; Bahn, 1988) ; trypsin (Fluka, Neu Ulm, Germany; Nietfeld et al., 1989) ; Kovacevic et al., 1997) ; pepsin (Dako, Hamburg, Germany; Bahn, 1988) , proteinase K and formic acid (Merck, Darmstadt, Germany; Hardt et al., 2000) ; and microwave pre-treatment (Kahveci et al., 2003) . Goat serum, diluted 1 in 5 in phosphate-bu¡ered saline (PBS), served as a blocking serum. Primary antibodies were, unless stated otherwise, diluted in PBS containing bovine serum albumin (BSA) 1%, and incubated overnight (12^16 h) at 4 1C ( Table 2) . Biotinylated goat-anti-mouse (gam-b), goatanti-rabbit (gar-b) and goat-anti-£uorescein-isothiocyanate (ga-FITC-b; directed against the FITCligated anti-feline infectious peritonitis [FIP] virus antibody) antibodies served as secondary antibodies (Vector Laboratories, Burlingame, CA, USA) and were all diluted 1 in 200 in PBS. Finally, sections were incubated with ABC (Vector) for 30 min at room temperature. Between the incubation steps, sections were thoroughly rinsed with PBS. Positive antigenâ ntibody reactions were ''visualized'' by incubation with 3,3 0 -diaminobenzidine-tetrahydrochloride (DAB) with H 2 O 2 0.5% for 10 min followed by mild counterstaining with haematoxylin. Sections were mounted with Roti-Histokit (Carl Roth KG, Karlsruhe, Germany) under coverslips. In control sections the primary and secondary antibodies were replaced by PBS, control serum or isotype-speci¢c antibodies directed against irrelevant antigens.

Toy breeds constituted 27% (14) ). Animals aged less than 1 year accounted for 38% of a¡ected dogs,37% were1^5 years old, and 21% were aged45 years. In 4% of dogs the age was unknown. The domestic shorthair breed accounted for 52% of affected cats. In 11 cats the breed was unknown. Cats less than 1 year of age accounted for 27%, 26% were aged 1^5 years, and 26% were45 years old. In 21%, the age was unknown. The sexes were represented equally in both species.

Neurological signs, including ataxia, seizures and central blindness, were reported in 35 dogs (68%). Nine dogs (17%) showed other symptoms such as vomiting and diarrhoea but lacked neurological signs. One dog died spontaneously and one under anaesthesia during routine surgery, no clinical signs having been reported before death. In seven dogs no clinical data were available.

Neurological signs were reported in 17 cats (52%). Thirteen (39%) showed gastrointestinal or respiratory signs but not neurological signs. In three cats, one of which died spontaneously, no clinical data were available.

Lymphohistiocytic, perivascular in¢ltrates were found in 40 dogs (75%) and 23 cats (70%). Predominantly granulomatous in¢ltrates were found in 10 dogs and two cats. Mixed in£ammatory in¢ltrates appeared more often in cats (six animals) than in dogs (two animals). Two cats and one dog exhibited pyogranulomatous in£ammation. In 39 dogs (74%), encephalitis was the most prominent ¢nding. In 13 dogs (25%), in¢ltrates were restricted to the meninx and a ganglioneuritis without encephalitis or meningitis was found in one animal. In 26 cats (79%), encephalitis was observed. In six cats (18%), in¢ltrates were restricted to the meninx and a chorioiditis without encephalitis was found in one animal. Six dogs showed, in addition to the in£ammatory changes in the CNS, mild-to-moderate vacuolation of the white matter of the cerebrum (three animals) or cerebellum (one animal) or both (two animals). Luxol-fast-blue staining revealed demyelination in two of these dogs. Only one of these six dogs showed an in-£ammatory in¢ltrate within the areas of vacuolation.

Two cats showed in addition to the in£ammatory changes in the CNS, mild vacuolation of the white matter of the cerebellum (one animal; severe) or both cerebrum and cerebellum (one animal; mild). Luxol-fastblue staining did not reveal demyelination.

Of the nine dogs without neurological signs, one showed mild, focal meningitis and two showed mild, multifocal meningitis. Moderate in£ammation, consisting of multifocal or focal in¢ltrations in various brain regions (midbrain, cerebellum, meninges, choroid plexus) was observed in six dogs. In£ammatory changes were lymphohistiocytic (six cases), mixed (two cases) or granulomatous (one case).

The 13 cats without neurological signs showed mild, multifocal in£ammation of the meninx (two cases), meninx and cerebellum (two), meninx and cerebrum (one), meninx, cerebrum and brainstem (one), meninx 

8000 (PBS contained 20% NSS but no BSA), adsorbed to rat brain powder

Microwave gam-b Anti-prion protein Sc 400 (PBS contained 10% goat serum but no BSA); no further blocking with goat serum necessary Formic acid, proteinase K, microwave

Anti-E. coli 1600 None gar-b NSS, normal swine serum; TUF, target unmasking £uid; gam-b, biotinylated goat-anti-mouse antibody; gar-b, biotinylated goat-anti-rabbit antibody; ga-FITC-b, biotinylated goat-anti-FITC antibody (see Materials and Methods). Ã Against agents listed inTable 1. y In phosphate-bu¡ered saline (PBS) containing bovine serum albumin (BSA) 1%. and brainstem (one) or choroid plexus (one). Also seen were mild-to-moderate in£ammation of the brainstem (one) and of the meninx and cerebral cortex in addition (one). One cat showed moderate, focal meningitis, one a moderate multifocal meningoencephalitis and one a severe, generalized choroiditis. The type of in£ammation was lymphohistiocytic in six cases, mixed in three, granulomatous in two, and pyogranulomatous in two. The gastrointestinal tissue examined in one dog, and the myocardial tissue examined in one dog and two cats, showed no signi¢cant histological changes.

In 14 dogs (26%) and 13 cats (39%), viral or bacterial antigens were detected by IHC (Table 3) , two dogs and one cat giving positive results for two infectious agents simultaneously. Control sections containing the appropriate antigens gave positive signals, in contrast to negative controls. Special histochemical stains invariably gave negative results.

In12 of the14 positive dogs and nine of the13 positive cats, reactions were found only in CNS regions in¢ltrated with in£ammatory cells. The remaining two dogs and four cats showed immunoreactivity only in CNS regions without in£ammatory in¢ltration.

Of the 14 positive dogs,12 (94%), had shown neurological signs, almost always said to be severe. It was not possible, however, to relate the histological or IHC results to the clinical signs. One dog positive for EMCV antigen did not show neurological signs, and for one dog positive for WNV antigen no clinical data were available.

Of the 13 positive cats, eight had shown neurological signs (severe in three cases). In the remaining ¢ve cats, no statement regarding the severity of signs was recorded. Four cats with a positive immunoreaction showed no neurological signs. For one positive cat, no clinical data were available.

In a 2-year-old male Canadian shepherd dog with diarrhoea and progressive convulsions, porcine herpesvirus-1 (PHV-1) antigen was demonstrated in neuronal perikarya of the gyrus parahippocampalis. No PHV-1 antigen was detected in the hippocampus, cerebellum, brain stem, spinal cord, remaining part of the cerebrum, or the gastrointestinal tract. Histologically, there was mild, lymphohistiocytic polioencephalitis in the gyrus parahippocampalis and occasional neuronal necrosis in the CA1 and CA2 regions of the hippocampus.

Parvovirus antigen was demonstrated in the CNS of ¢ve Greek hunting dog puppies from two litters originating from the same breeder, and in a 2-week-old cat. Clinically, the dogs had shown generalized tremor and jumping movements in the hind limbs, and the neurological signs in the cat were characterized by severe ataxia, opisthotonus and convulsions. Neither in the dogs nor in the cat were cerebellar hypoplasia or necrotic Purkinje cells observed. Histopathologically, all affected dogs showed mild to moderate lymphohistiocytic meningitis or leucoencephalitis (or both) as well as mild to moderate, and in one case severe, vacuolation in the white matter of cerebrum and cerebellum. Parvovirus antigen was demonstrated in periventricular cells resembling spongioblasts, in macrophages, microglia and astrocytes, and in cells of the outer granular layer of the cerebellum (Fig. 1) . The histopathological changes in the cat consisted of mild, lymphohistiocytic in£ammation of the meninges and cerebellum, without vacuolation. Parvovirus antigen was detected in the cytoplasm and nucleus of numerous small and large neurons, mainly in the granular layer and outer granular layer of the cerebellum, in the grey matter of the spinal cord, and multifocally in most other brain regions (Fig. 2) . In addition, parvovirus antigen was observed in macrophages, microglia, astrocytes and ependymal cells.

FIP virus antigen was detected in three cats aged 6,18 and 36 months.Two a¡ected cats had shown increasing apathy and recurrent fever attacks (one animal) or diarrhoea and dyspnoea (one). No clinical data were available for the third cat. Viral antigen was demonstrated in the cytoplasm of macrophages within the in-£amed brain regions in all three cases. Histologically, lesions consisted of pyogranulomatous and mixed in¢ltrates in the plexus chorioideus and the meninges. The third cat showed moderate lymphohistiocytic leucoencephalitis in the caudal regions of the cerebrum. 

Weak immunolabelling for feline leukaemia virus antigen was seen in microglia and astrocytes in the hypothalamus of a 9.5-year-old European shorthair cat with progressive apathy and increased salivation (Fig. 3) . Histopathological changes consisted of mild multifocal lymphohistiocytic meningitis and severe satellitosis in the cerebral cortex. No lymphoid tissues or other organs were available for further investigations.

Five dogs (8.9%) and four cats (11.8%) showed immunoreactivity for West Nile virus (WNV) antigen. In one dog, canine parain£uenza virus (CPIV) antigen was also detected in the same brain region (see also below). One of these dogs, and one cat also gave positive reactions with EMCV-speci¢c antibodies (see also below). In four of the ¢ve dogs, severe neurological signs, including ataxia, convulsions and central blindness, had been recorded. No clinical data were avail-able for the ¢fth dog. All four cats had shown neurological signs, reported in one case to consist of severe circling, hypermetria and blindness. In four of the ¢ve dogs and two of the four cats a moderate to severe meningoencephalitis a¡ecting the grey and white matter was observed; this varied from granulomatous, pyogranulomatous, lymphohistiocytic to ¢brinopurulent, occasionally associated with neuronal necrosis or malacia. In the remaining dog, in£ammation was restricted to the grey matter of the cerebrum and cerebellum, being associated with severe malacia in the hippocampus. One cat showed mild focal, lymphocytic polioencephalitis and moderate focal, lymphohistiocytic meningitis, as well as severe vacuolation of the cerebral white matter and severe generalized neuronal necrosis. In another cat, severe, focal, ¢brinopurulent meningitis and severe, acute neuronal necrosis in the hippocampus were observed.

WNV immunolabelling was seen in the cytoplasm of various types of neurons, macrophages, astrocytes and microglia (Fig. 4) . Strong immunolabelling of neutrophils was observed in two cats with pyogranulomatous in£ammation (Fig. 5) . These immunohistochemical reactions were in general more intense with the anti-NSP-1WNVantibody than with the anti-MEP-E WNVantibody. In some cases, immunoreactivity was observed with only one of these two WNV monoclonal antibodies. One dog showed immunoreactivity in pyramidal cells of the CA1 and CA2 sector of the hippocampus, and another in the frontal cortex, respectively. A positive reaction in numerous macrophages and microglial cells in the midbrain, pons and frontal cerebrum was detected only with the anti-NSP-1 antibody. One dog showed WNV immunolabelling in numerous macrophages and microglial cells, and pyramidal cells in the cerebrum, hippocampus and midbrain, but only with the anti-NSP-1 antibody. Another dog, in contrast, showed immunoreactivity in a few neurons and macrophages in an in£amed part of the midbrain, but only with the anti-MEP-E antibody. One cat showed immunolabelling with both WNV antibodies in numerous neutrophils and occasional macrophages in the plexus chorioideus and laterally to the third ventricle. In this cat, small neurons, macrophages and microglial cells in the same brain region reacted only with the anti-NSP-1 antibody. In another cat, astrocytes and perivascular macrophages in the lobus parietalis reacted with both antibodies, whereas the anti-NSP-1 antibody additionally labelled pyramidal cells in the same brain region. In one cat, astrocytes and oligodendrocytes of the medulla oblongata displayed more abundant positive signals with the anti-NSP-1 antibody than with anti-MEP-E. Additionally, in this animal, the anti-NSP-1 antibody labelled large neurons in the medulla oblongata which failed to react with the anti-MEP-E antibody. Canine parain£uenza virus (CPIV) antigen was detected in the brain of a 9-year-old male collie that was also positive forWest Nile virus antigen (see alsoWNV, above). This dog had exhibited changed behaviour and was generally weak. Histopathologically, severe periventricular granulomatous meningoencephalitis was found. Viral antigen was demonstrated in the cytoplasm of occasional macrophages in the hypothalamus (Fig. 6) .

IHC revealed encephalomyocarditis virus (EMCV) antigen in the brains of four dogs (7.1%) and four cats (11.8%). In one dog and one cat, WNV-antigen was detected in addition (see also WNV, above). The dogs were aged 0.3^8 years and the cats 0.2^10 years. Two domestic shorthairs, one Persian and one cat of unknown breed as well as a Landseer, a West Highland white terrier, a Bordeaux Dogge and an Entlebucher Sennenhund were a¡ected. A granulomatous immune response was found in three of the four dogs. The remaining dog displayed a lymphohistiocytic chorioiditis and meningitis. In¢ltration with macrophages, neutrophils, lymphocytes and plasma cells or pyogranulomatous in£ammation was present in two of the four cats, and lymphohistiocytic in£ammation in the remaining two cats. In three of these four dogs, severe neurological signs had been recorded; the fourth dog had shown dyspnoea and anaemia but no neurological signs. In three of the cats, neurological signs were recorded without reference to their severity. The fourth cat showed respiratory signs only. The eight animals showed di¡erent patterns of immunoreactivity. DAB precipitates were observed in the cytoplasm of perivascular and parenchymal macrophages, in the perikarya and processes of various neurons distant Non-suppurative Meningoencephalitis from or within the in£ammatory lesions (Fig.7) , and in periventricular and submeningeal astrocytic foot processes (Fig. 8) . In one dog and two cats, vessel-associated cells, probably pericytes or endothelial cells, showed immunolabelling (Fig. 9) . Seven of the eight animals showed immunolabelling with the EMCV-speci-¢c monoclonal antibody (mab) 4F3; mab 3E5 gave results in selected cases only. One cat which was negative with the 4F3 antibody gave EMCV-speci¢c reactions with mab 3E5. In one dog the results with mab 4F3 were con¢rmed with the mab 3E5. The mab 4E4, which was applied only in one cat, con¢rmed the reactions observed with the 4F3 antibody in numerous cells of the medulla oblongata and in perivascular macrophages and occasional neurons of the mediocaudal thalamus. The histologically intact myocardial tissue available from one dog and two cats was not immunolabelled with the mab 4F3.

E. coli antigen was demonstrated in the brain of a 5-month old male cat. Histopathologically, mild to moderate, multifocal, perivascular in¢ltration with lymphocytes, plasma cells, macrophages and neutrophilic granulocytes was observed in the cerebellum and meninges. Moderate numbers of bacterial emboli in various vessels were visible in HE-stained sections. E. coli antigen was demonstrated immunohistochemically in the cytoplasm of numerous perivascular, meningeal and periventricular macrophages, and in intravascular bacteria.

Cases remaining unclear included 39 (74%) dogs and 20 (61%) cats, no infectious agent being identi¢ed as the underlying cause of the observed non-suppurative meningoencephalitis.

In the present study, CNS tissue of 53 dogs and 33 cats with non-suppurative meningoencephalitis of unknown cause was investigated for the presence of 18 different infectious agents. Former studies either investigated at most six agents or were based on cases selected only on the presence of neurological signs (Tipold, 1995; Quesnel et al., 1997; Bradshaw et al., 2004; K. Melzer, personal communication) .

As in other studies (Sorjonen, 1992; Tipold, 1995) , no speci¢c canine breed predisposition was observed. Cases were seen more often in toy breeds than in other breeds, but this may merely have re£ected the normal distribution of dog breeds in Germany. So far, no breed predisposition for non-suppurative meningoencephalitis has been described in respect of the cat (Sorjonen, 1992) . In the present study, domestic shorthair cats accounted for more than half of the feline cases, probably due to the normal feline breed distribution in Germany.

The CNS of the majority of dogs (40/53) and cats (23/ 33) examined showed lymphohistiocytic perivascular in¢ltrates. This is generally suggestive of a viral aetiology (Summers et al., 1995) but has also been reported following hypoxic or toxic damage, which also sometimes produces demyelinating lesions (Braund, 1980; Lee et al., 2002) . Granulomatous CNS in¢ltrates, observed in 10 dogs and two cats, are often considered to be associated with fungal, parasitic or bacterial infections. Fungal infections occur mainly in warmer climates (Gerds-Grogan and Dayrell-Hart, 1997 ) and, like parasitic infections, were not detected in the present study. In the 10 dogs referred to above, granulomatous meningoencephalitis (GME) was suggested by the histopathological ¢ndings and distribution of lesions. In six of these cases, toy breeds were a¡ected (twoWest Highland white terriers, two chihuahuas, a shih tzu and a rehpinscher). Interestingly, three of the a¡ected dogs belonged to large breeds (Landseer, Bordeaux Dogge and collie), in which GME is only rarely reported (Braund, 1985) . GME has been thought to be associated with viral infections including canine distemper, but no viral agent has been demonstrated in the CNS so far (Thomas and Eger,1989; Kipar et al.,1998) . E. coli antigen, as found in the brains of beagles with granulomatous leptomeningitis (Maeda et al., 1993) or in association with human cases of granulomatous encephalitis (Rickert et al., 2000) , was not observed in the present cases with suspected GME. The cat with abundant E. coli antigen in the brain in the present study showed perivascular in-¢ltrations, indicating septicaemia.

Pyogranulomatous and mixed in£ammatory in¢ltration in the CNS occurred less often in dogs than in cats. In cats, this type of change is reminiscent of FIP (Rand et al.,1994) . In the present study, FIP virus antigen was also detected in a cat showing lymphohistiocytic leucoencephalitis, a rare consequence of FIP (Summers et al.,1995; Braund, 2001) . In dogs, pyogranulomatous or mixed in£ammatory changes in the CNS are occasionally described, but no infectious agents have so far been detected (Meric,1988) .

It cannot be ruled out that aetiologically undetermined feline cases were due to feline immunode¢ciency virus (FIV), which leads to lymphocytic encephalitis in 30% of naturally infected cats (Gunn-Moore et al., 1996) . Only ¢ve of the 33 cats had been examined serologically for FIV, a positive result having being obtained in one animal.

In a Canadian shepherd dog, porcine herpesvirus 1 (PHV 1) antigen was detected in neurons of the gyrus parahippocampalis. Due to a retrograde neural route of infection, PHV 1 antigen usually occurs in the brain stem or spinal cord ganglions (Quiroga et al., 1998; Honavar and Meldrum, 2002) . The dog in this study showed diarrhoea for 3 weeks, reminiscent of the rare alimentary form of Aujeszky disease (Pensaert and Maes, 1987) ; however, no PHV 1 antigen was detected in the gastrointestinal tract. Only later did the animal develop neurological signs, and an unusual portal of entry of the virus to the brain remains a possibility.

Neither the ¢ve dogs nor the cat with parvovirus infection of the brain showed typical CNS manifestations, such as cerebellar hypoplasia or death of Purkinje cells (Sharp et al., 1999) , or encephalomalacia (Johnson and Castro, 1984; Agungpriyono et al., 1999) . The ¢ndings indicated, however, a potential association between parvovirus and spongiform lesions in the CNS.

In chronic feline leukaemia virus (FeLV) infection, lymphocytic in¢ltration of peripheral nerves of the spinal cord has been reported occasionally (Pedersen, 1987) . In the brain, however, FeLV infection has been associated only with degenerative CNS disease (Carmichael et al., 2002) . In one cat, FeLV infection of microglia and astrocytes was detected. Histologically, this animal showed mild, multifocal, lymphohistiocytic meningitis. Since FeLV regularly persists for years in lymphocytes and macrophages (Rohn and Overbaugh,1999) , a persistent infection of glial cells seemed possible but unfortunately no lymphoid tissue, including bone marrow (in which FeLV-antigen can typically be demonstrated), was available.

Surprisingly, ¢ve (9.4%) dogs and four (12%) cats showed immunolabelling for WNV. Histopathologically, these animals showed mild to moderate meningoencephalitis in the grey and white matter, mostly with granulomatous or pyogranulomatous in£ammation. In contrast, previous reports of WNV infection in birds and in mammals indicated that the grey matter was mainly a¡ected and that the histopathology was characterized by lymphoplasmahistiocytic in£ammation with small numbers of neutrophils (Steele et al., 2000; Cantile et al., 2001; Buchweitz et al., 2003; Kelly et al., 2003; Lichtensteiger et al., 2003) . Polyclonal WNV-speci¢c antibodies used for immunolabelling often cross-react with other £aviviruses (Steele et al., 2000; Kelly et al., 2003; Chvala et al., 2004) . The monoclonal antibodies speci¢c for the major envelope protein E (anti-MEP) and non-structural protein 1 (anti-NSP-1) used in this study (Tables 1 and 2 ) crossreact with Kunjin virus, also a member of the Flaviviridae (Scherret et al., 2001) . Presumably, the anti-NSP-1 antibody is more speci¢c than the anti-MEP antibody (Hall, 2000) . This might explain its stronger immunoreactivity in the present study and might support the identi¢cation of a WNV infection. The a¡ected animals did not react positively for tick-borne encephalitis (TBE) virus antigen with a polyclonal speci¢c antibody that may cross-react with other £aviviruses (H. Weissenbo º ck, personal communication). However, it is still possible that the positive WNV reaction was due to infection with other cross-reactive agents or represented molecular mimicry of host-derived antigens (Oldstone, 1998) . The latter possibility receives some support from studies indicating that dogs and cats can be infected with WNV but usually develop only a low viraemia, without clinical signs or histopathological changes in the brain (Blackburn et al.,1989; Lichtensteiger et al., 2003; Austgen et al., 2004) . Furthermore, no evidence of WNV infections in domestic animals has so far been detected in Germany. Therefore, the signi¢cance of the present ¢ndings requires further studies.

Natural infections of dogs with canine parain£uenza virus (CPIV) are typically associated with hydrocephalus and necrotizing periventricular encephalitis, but neither viral antigen nor RNA has been detected so far in spontaneous cases (BaumgÌrtner et al., 1982b; Vieler et al.,1994; Cantile et al.,1997) . After experimental infection with CPIV, viral antigen was detected in ependymal cells and neurons and in macrophages, in ferrets and dogs respectively (BaumgÌrtner et al., 1982a (BaumgÌrtner et al., , b, 1989a W. BaumgÌrtner, unpublished) . CPIV infection of brain macrophages was also observed in the present study in a collie with granulomatous encephalitis characteristic of GME. Interestingly, this dog also showed immunolabelling of WNV antigen. It remains unclear, therefore, whether the detection of CPIVantigen represented a true infection, or whether immunoreactivity was due to a cross-reaction of the antibody with degraded cellular antigens, or to molecular mimicry (Oldstone,1998) .

A further surprising ¢nding was the detection of EMCVantigen in the CNS of four dogs and four cats. EMCV-speci¢c antibodies have been detected in primates, mice, rats, horses and elephants in various parts of the world, the rat being the natural host (Nowotny et al.,1993; Nowotny,1996; Psalla et al., 2006) . In Austria, a low seroprevalence has been reported in cats (Nowotny,1996) , but not dogs. In Germany, EMCV-associated non-suppurative meningoencephalitis is reported occasionally in man, but the virus has been isolated from the CNS in only a few cases (Nowotny et al., 1993; LaRue et al., 2003) . The histopathological changes reported here varied (neuronal necrosis, mild lymphohistiocytic meningitis, severe generalized granulomatous or pyogranulomatous meningoencephalitis). In pigs with natural EMCV infections, lymphohistiocytic in¢ltrates in the CNS have been described (Gainer et al., 1968) . Experimentally infected mice and pigs also develop lymphohistiocytic, perivascular in¢ltrates in the CNS, poliomeningoencephalomyelitis being the predominant feature in mice (Topham et al., 1991; LaRue et al., 2003) . In the present study, EMCV immunolabelling was observed in perikarya and processes of neurons in some animals, indicating neurotropism similar to that observed in experimental EMCV infection. The EMCV-positive cells, associated with cerebral vessels, may have been endothelial cells or pericytes. Neither cell type has been reported to be susceptible to EMCV, but endothelial and mesenchymal cells in organs other than the CNS are regularly infected with EMCV, as shown by Papaioannou et al. (2003) in pigs. The detection of EMCV antigen in macrophages in the CNS of dogs and cats may support their role in viral replication and distribution, as proposed by Papaioannou et al. (2003) . In natural EMCV infection of pigs, interstitial myocarditis and myocardial degeneration represent the main features (Papaioannou et al., 2003) . Neither change was observed in any of the present cases, but myocardial tissue was available from only one dog and two cats.

Despite the broad spectrum of antibodies used, the aetiology of the CNS lesions could be determined in only 26% and 39% of the cases in dogs and cats, respectively. Due to the retrospective character of the study, a standardized clinical and neurological investigation and serum and CSF laboratory procedures were not possible. In the remaining cases, infection cannot be ruled out. Other agents that might have caused the observed histopathological changes in dogs include Ehrlichia spp. (Glaus and Jaggy, 1992; Maretzki et al., 1994) , Leptospira spp. (Rentko et al., 1992) and Borrelia spp. (Mandel et al.,1993; Chang et al., 2001) , and in cats Bartonella henselae (Breitschwerdt et al., 1999) cannot be excluded. However, the high percentage of cases in which no infectious agent was identi¢ed suggests that non-infectious causes are more common than anticipated. Various factors, ranging from autoimmune processes to intoxication, have already been suggested as causes of non-suppurative changes in the CNS (Kipar et al.,1998; , Lee et al., 2002 .

The immunohistochemical reactions for WNV, CPIVand EMCV, which were unexpected, were possibly due to molecular mimicry, a phenomenon reported for various paramyxoviruses and forJapanese encephalitis virus (JPEV; Shesberadaran and Norrby, 1984) , which belongs to the Flaviviridae. In most of the cases showing these unexpected reactions the histopathological changes were severe and often resembled GME. Autoimmune mechanisms have already been discussed as a possible cause of canine GME and pug dog encephalitis (Kipar et al., 1998; . Other factors that may have played a role in the ¢ndings described include the following: epitope spreading Vanderlugt et al., 1998) ; hereditary factors, as already suspected in pug dog encephalitis and non-suppurative meningoencephalitis in greyhounds (Hinrichs et al., 1996; Callanan et al., 2002) ; intoxication (Lee et al., 2002) ; increased permeability of the blood-brain barrier in systemic illness, due to prostaglandins and interleukin (IL)-1, IL-6 or tumour necrosis factor (TNF)-a, leading to in¢ltration of the CNS by in£ammatory cells (Reiss et al., 2000) . In addition, it should be mentioned that, unlike paraneoplastic encephalitis, which has been described in man but not in dogs or cats (Scaravilli et al., 1999) , paraneoplastic polyneuropathy has been described in dogs (Braund, 1990; Wagner, 2002) . As tumours in the CNS or other organs were present in only one of the cases examined, paraneoplastic encephalitis seemed unlikely to have played any role in the ¢ndings.

In conclusion, in a high proportion of dogs and cats with non-suppurative meningoencephalitis, immunohistochemical examination with antibodies against 18 di¡erent infectious agents failed to reveal a cause. The signi¢cance of positive immunoreactions obtained with WNV and EMCV antibodies requires further investigation. In addition, primary or virus-triggered secondary immune-mediated mechanisms should be considered in many in£ammatory CNS disorders of dogs and cats.

Subacute massive necrotizing myocarditis by canine parvovirus type 2 infection with di¡use leukoencephalomalacia in a puppy

Experimental infection of dogs and cats with West Nile virus. Emerging Infectious Diseases

The proteolytic pretreatment of formalin-¢xed tissue in immunohistochemical diagnosis. Acta Histochemica, Supplements

Canine parain£uenza virus-induced encephalitis in ferrets

Acute encephalitis and hydrocephalus in dogs caused by canine parain£uenza virus

Naturally occurring canine distemper virus encephalitis: distribution and expression of viral polypeptides in nervous tissues

Chlamydia pneumoniae in koronarem Plaquegewebe: Vermehrter Nachweis bei akutem Koronarsyndrom

Susceptibility of dogs to West Nile virus: a survey and pathogenicity trial

The functional signi¢cance of epitope spreading and its regulation by co-stimulatory molecules

A retrospective study of 238 cases of neurological disorders of the cat

Granulomatous meningoencephalomyelitis

Remote e¡ects of cancer on the nervous system

In£ammatory diseases of the central nervous system

Peripheral neuropathy associated with malignant neoplasms in dogs

Fading kitten syndrome and neonatal isoerythrolysis

reverse transcriptase-polymerase chain reaction, and immunohistochemical detection of West Nile virus in a clinically a¡ected dog

A novel non-suppurative meningoencephalitis in young greyhounds in Ireland

Hydrocephalus with periventricular encephalitis in the dog

Pathologic and immunhistochemical ¢ndings in naturally occurring West Nile infection in horses

Feline leukaemia virus-associated myelopathy in cat

Experimental induction of chronic borreliosis in adult dogs exposed to Borrelia burgdorferi-infected ticks and treated with dexamethasone

Pathology and viral distribution in fatal Usutu virus infections in birds from the

Intranasal infection of ferrets (Mustela putorius furo) with canine parain£uenza virus

Localization of neutralizing regions of the envelope gene of feline leukaemia virus by using anti-synthetic peptide antibodies

Zur Diagnostik der Aujeszkyschen Krankheitçeine vergleichende Untersuchung immunhistologischer und virologisch-kultureller Nachweismethoden

High mortalitiy in a Florida swine herd infected with the encephalomyocarditis virus. An accompanying epizootiological survey

Feline cryptococcosis: a retrospective evaluation

Ehrlichiose beim Hund: Literaturu º bersicht und Fallbeschreibung

Encephalitis associated with giant cells in a cat with naturally occurring feline immunode¢-ciency virus infection demonstrated by in situ -hybridization

The emergence of West Nile virus: the Australian connection

A comparative study of immunohistochemical methods for detecting abnormal prion protein with monoclonal and polyclonal antibodies

Simultaneous central nervous system reticulosis in two related Afghan hounds. Compendium on Continuing Education for the PracticingVeterinarian

Comparison of di¡erent methods of diagnosing Borna disease in horses post mortem

Ein Fall von nekrotisierender Meningoencephalitis beim Mops (pug dog encephalitisçPDE) (In German

Epilepsy

Use of avidinbiotin-peroxidase complex, ABC, in immunoperoxidase techniques

Isolation of canine parvovirus from a dog brain with severe necrotizing vasculitis and encephalomalacia

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin ¢xed

The histopathology of rectosigmoid biopsies from adults with bloody diarrhea due to verotoxin-producing

The neuropathology of West Nile virus meningoencephalitis

Immunohistochemical characterization of in£ammatory cells in brains of dogs with granulomatous meningoencephalomyelitis

Expression of viral proteins in feline leukaemia virus-associated enteritis

West Nile viral encephalitis. Revue Scienti¢-que et Technique (International O⁄ce of Epizootics)

Immunohistochemical diagnosis of feline leukaemia virus infection in formalin-¢xed tissue

A wild-type porcine encephalomyocarditis virus containing a short poly C tract is pathogenic to mice, pigs and cynomolgus macaques

Comparative immunological characterization of type-speci¢c and conserved B-cell epitopes of pinniped, felid and canid herpesviruses

Ibuprofen-induced aseptic meningoencephalitis

West Nile encephalitis and myocarditis in wolf and dog

Staggering disease in cats: isolation and characterizaton of the feline borna disease virus

In£ammatory disease of the central nervous system

Granulomatous leptomeningitis in beagle dogs

Intrathecal production of Borrelia burgdorferi-speci¢c antibodies in a dog with central nervous system lyme disease. Compendium on Continuing Education for the Practicing Veterinarian

Granulocytic ehrlichiosis and meningitis in a dog

Canine meningitisça changing emphasis

Rabies-like inclusions in dogs

Serological studies of domestic cats for potential human pathogenic virus infections from wild rodents

Encephalomyocarditis-Virusinfektion beim

German.). Wiener TierÌrztliche Monatsschrift

Description of feline meningoencephalomyelitis (''staggering disease'') and studies of its aetiology

Molecular mimicry and immunemediated diseases

Pathogenesis of encephalomyocarditis virus (EMCV) infection in piglets during the viraemia phase: a histopathological, immunohistochemical and virological study

Feline leukaemia virus

Pseudorabies virus (Aujeszky 0 s disease)

Pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats

Diagnostic evaluation of cats with seizure disorders: 30 cases

Diagnosis of Aujeszky 0 s disease virus infections in dogs by use of immunohistochemistry and in-situ-hybridization

Clinical, cerebrospinal £uid and histological data from twenty-seven cats with primary in£ammatory disease of the central nervous system

Innate immune responses in viral encephalitis

Canine leptospirosis: a retrospective study of 17 cases

Pathogenic feline retroviruses: feline leukaemia virus and feline immunode¢-ciency virus

Mikroskopische Technik, 17th Edit

The neuropathology of paraneoplastic syndromes

Hydranencephaly and cerebellar hypoplasia in two kittens attributed to intrauterine parvovirus infection

Three monoclonal antibodies against measles virus F protein cross-react with cellular stress proteins

Myelitis and meningitis

Pathology of fatal West Nile virus infections in native and exotic birds during the

Susceptibility of primary cell culture neurones from rats of di¡erent ages to encephalomyocarditis, EMC, virus infection

Granulomatous meningoencephalomyelitis in 21 dogs

Diagnosis of in£ammatory and infectious diseases of the central nervous system in dogs: a retrospective study

Indirect role of Tcells in development of polioencephalitis and encephalomyelitis induced by encephalomyocarditis virus

The functional signi¢cance of epitope spreading and its regulation by co-stimulatory molecules

Polioencephalomyelitis in cats

Isolation of parain£uenzavirus type 2 from the prostatic £uid of a dog

Immunohistochemical detection of encephalomyocarditis virus (EMCV) both in formalin and ethanol-¢xed, para⁄n-embedded tissues of pigs

Identi¢cation of CD4+ and CD8+ T cell subsets and B cells in the brain of dogs with spontaneous acute, subacute, and chronic-demyelinating distemper encephalitis

Glycoprotein speci¢c immune response in canine herpesvirus infection

For providng antibodies, the authors thank: Prof. Haas, Institute of Virology, University of Veterinary Medicine Hannover, Germany, Prof. Richt, National Animal Disease Center, Ames, Iowa, USA; Dr Eskens, VeterinÌr-Untersuchungsamt Mittelhessen; Prof. Holzmann, Institute of Virology, University of Vienna; Prof. Groschup, Bundesforschungsanstalt fu º rViruskrankheiten derTiere, Insel Riems; Dr Randall, Department of Biochemistry and Microbiology, University of St Andrews, UK; and Dr O º rvell, Huddinge Hospital, Huddinge, Sweden. Thanks are also due to Mrs Petra Gru º nig and Mrs Danuta Waschke for excellent technical support.