**3. Results**

32 Non-Flavivirus Encephalitis

*Immunohistochemical (IHC) examination*: on histological sections with suspected lesions, specific antigens for *Listeria* spp*. Encephalitozoon cuniculi*, *Streptococcus suis*, *Neospora caninum*, and *Toxoplasma gondii* were investigated. Antibodies from commercially available kits were used according to the methods specific for the antigen. Deparaffinized and rehydrated sections were subjected to different types of unmasking according to the protocols: microwave (750 W), trypsin (0.1% with 0.1% Ca carbonate) or in a bain-marie (citrate buffer pH 6.1 to variable temperature). After rinsing, the sections were incubated for 4-12 h at 37°C or overnight at 4°C with specific monoclonal antibodies diluted (1:50; 1:500; 1:1000) in PBS. Subsequent antibody detection was carried out using biotinylated goat antimouse or anti horse secondary antibody diluted (1:200-1:1000) in PBS, for 20 min at room temperature, followed by the avidina-biotin-peroxidase complex. (Vectastain ABC kit, Vector Laboratories). Immunoreactivity was visualized using 3,3'-diaminobenzidine as chromogen; the sections were counterstained with Mayer's haematoxylin counterstain. *Cultural examination*: bacteria isolation provides a first incubation at 35-37° C for 24-48 h on selective (Demi-Fraser; Oxford agar; Mac Conkey) and non-selective culture medium (blood agar with 5% sheep blood). Biochemical identification of the isolated strain was carried out

*Biomolecular examination*: frozen brain biopsies were individually macerated in a laminar flow bench and total nucleic acids were purified using a NucliSENS® easyMAG® system (bioMérieux, Inc., Durham, NC, USA) according to the manufacturer's instructions. Formalin-fixed paraffin-embedded samples were initially deparaffinizated using a xylenebased technique prior to nucleic acid extraction. All samples were tested for cellular adequacy and absence of PCR inhibitors by PCR amplification of the b-actin gene DNA (for DNA extraction) or cDNA (for RNA extraction) as previously described (Salata et al., 2009). The samples were tested by real-time RT-PCR for West Nile Virus (WNV) and enterovirus-RNA detection using the oligonucleotide primers and TaqMan probe targeting the WNV E gene (Lanciotti et al. , 000) or the enteroviral genome 5' untranscribed region (Donaldson et al., 2002). In the procedure used, the nucleic acid (about 60 ng) was combined with Superscript® One Step RT-PCR System reagents (Invitrogen Ltd, Paisley, UK), primers and probe, reaching a total reaction volume of 20 μl, and amplified in a LightCycler® 2.0 real-

Real-time PCR assays were used to test for the presence of *Toxoplasma gondii* (Lin et al. , 2000), *Borrelia burgdorferi* (Exner et al., 2003), *Listeria* spp. and *L. monocytogenes* (Rodríguez-Lázaro et al. ,2004), and *Chlamydia* spp. (Yang et al., 2006) with some modifications. Briefly, the extracted nucleic acid (about 60 ng) was assayed with an ABI PRISM 7700 sequence detector system (Applied Biosystems, Foster City, CA, USA) in 25 μl of a PCR mixture containing 12.5 μl TaqMan universal master mix, 15 pmol of each primer, and 10 pmol of the

End-point PCR assays were used to assay all samples for Borna virus (Cotto et al., 2003), tick-borne encephalitis virus (Puchhammer-Stoeckl et al., 1995), herpesvirus (Rose et al. , 1997) and fungi (Sutar et al., 2004). Reverse transcription, if necessary, was performed as described by Bergonzini et al., 2009. Thermal cycling conditions were: one cycle at 95°C for 10min and 40 cycles at 95°C for 30 s; 50-60°C for 45 s; 72°C for 1 min; and an additional cycle

To detect herpesvirus, a set of degenerate PCR primers targeting the highly conserved DNA polymerase genes of the Herpesvirus family was used, while in the case of fungi the rDNA internal transcribed spacer sequences were targeted by primers. Subsequently, herpesvirus

on pure culture with an API Rapid system.

time PCR System (Roche Diagnostics S.p.A., Monza, Italy).

probe under standard amplification conditions.

at 72°C for 10 min.

In 660 animals (42%), traumatic, vascular, congenital, muscular and articular diseases were diagnosed by clinical examination; no CNS involvement was found. In 353 subjects (23%), probable zoonoses were identified; 82 of these (73 dogs and 9 cats) showed neuropathological lesions associated with viral infections of unknown origin and therefore considered as "potential zoonoses" in the literature (Graph. 2). In 337 (22%) cases, pathologies not transmissible to humans were present: neoplasia, toxic-degenerative and bacterial diseases, distemper, FIV, FELV, FIP, IBR, CAEV Visna, border disease, etc., and therefore were not considered in this study. In 194 cases (12%), no diagnosis was established because the case history was incomplete and/or tissues were unsuitable for biomolecular investigations. Sixteen animals (1%) showed no lesions (Graph. 3). Overall, 16 zoonoses were detected in 271 animals of different species (Table 1).

**Encephalitozoonosis** - In 110 (31.5%) rabbits, *Encephalitozoon cuniculi* was isolated. The clinical signs were variable, reflecting the site of lesions in the brain: convulsions, discoordination, paresis or paralysis and stiff neck were reported. The thalamus and hippocampus presented with more severe damage. In all animals there was a granulomatous meningo-encephalitis characterized by perivascular cuffs and focal granulomas composed of epithelioid histiocytes and lymphocytes, some of which showed a centre composed of amorphous eosinophilic material (Fig. 1). In all, 78% of these subjects were serologically positive for *Encephalitozoon cuniculi*; and in some cases IHC and Ziehl-Neelsen stain highlighted individual or aggregates of parasites within the granulomas, thus confirming the diagnosis (Figs. 2 and 3).

**Listeriosis** - In 47 (13.5%) cases (33 cattle and 14 sheep) *Listeria* spp. was isolated. Clinically, the animals showed depression, fever, paralysis, twisted neck, tremors, ataxia, motion

Zoonoses Surveillance in Italy (2000-2009):

Table 1. Zoonoses diagnosed in the period of study

Investigation on Animals with Neurological Symptoms 35

\* GME: Granulomatous Meningoencephalitis; SRMA: Steroid-responsive meningitis-arteritis; NEM/NLE: Encephalo-mielitis/Leuco-encephalitis necrotizing; EME: Eosinophilic maningoencephalitis; NSME: Non suppurative meningo-encephalitis; PVE: Periventricular encephalitis ; FPE: Feline polio-encephalomielitis ; MUE: Meningoencephalomyelitis of undetermined etiology

Graph. 2. Graphic representation of diseases of **"unknown aetiology"**.

Graph. 3. Graphic representation of results obtained in this study


Table 1. Zoonoses diagnosed in the period of study

\* GME: Granulomatous Meningoencephalitis; SRMA: Steroid-responsive meningitis-arteritis; NEM/NLE: Encephalo-mielitis/Leuco-encephalitis necrotizing; EME: Eosinophilic maningoencephalitis; NSME: Non suppurative meningo-encephalitis; PVE: Periventricular encephalitis ; FPE:

Feline polio-encephalomielitis ; MUE: Meningoencephalomyelitis of undetermined etiology

42%

No CNS involvement Probable zoonoses

No zoonoses Unsuitable No lesions

Graph. 2. Graphic representation of diseases of **"unknown aetiology"**.

23%

Graph. 3. Graphic representation of results obtained in this study

12% 1%

22%

0

5

10

15

20

Zoonoses Surveillance in Italy (2000-2009):

Fig. 5. Cow. *Listeria monocytogenes*: bacterial antigen within a microabscess (IHC, 40x).

was confirmed by bacterial culture.

protoscolices were visible.

Investigation on Animals with Neurological Symptoms 37

12 cases *S. suis* was found only at the level of the trachea and lungs, but only 6 of these had neuropathological lesions. IHC confirmed *S. suis* serotype 2 infection in 10 cases (Fig. 7).

**Colibacillosis** - In 21 (6%) cases, involving 15 calves, 1 lamb and 5 piglets, *E. coli* was isolated. All presented with hyperacute or acute septicaemia, depression, ataxic gait, opisthotonus and sudden death. The neuropathological lesions were similar in all cases and showed a massive suppurative meningo-encephalitis characterized by mononuclear cells and a high amount of neutrophils. There was also marked vasculitis throughout the brain; in some cases, abscess-like lesions were visible, predominantly in the brainstem. The diagnosis

**Coenurosis -** In 17 (5 %) cases (1 cattle and 16 sheep) larvae of the *Taenia multiceps* tapeworm were identified. Clinical signs and severity of the acute phase depended on the number of eggs ingested, the extent of inflammatory response, and the location of the parasite in the CNS. The clinical records reported weight loss, apathy, adynamia, tendency to isolation, depression, blindness, head tilt and ataxia. Histologically, focal haemorrhage or malacia caused by the migratory phase of the larval stage (acute stage), were observed at the level of the cerebral hemispheres or in the caudal brainstem. In the chronic phase, the visible cysts were surrounded by atrophic tissue. The diameter of the cysts, single or multiple, varied from 1 to 4.5 cm and appeared as an empty space surrounded by a granulomatous reaction composed of macrophages, giant cells, lymphocytes and plasma cells, and a few granulocytes. Outwardly, many macrophages, more or less extensive haemorrhage, fibrin or fibrin-purulent leptomeningitis and mononuclear perivascular cuffs were sometimes observed. In many cases it was possible to extract the parasitic cysts in which the cephalic

**West Nile encephalitis** - West Nile virus was isolated in 15 (4%) horses. The horses were 9 females and 6 males, 2 to 19 years of age, mostly trotters from the provinces of Ferrara and Bologna. The horses underwent detailed neurological examination, blood-biochemical and serological tests. The blood-biochemical tests showed no significant changes, except in 5 horses in which there were slight alterations of inflammatory proteins in the acute phase. Three horses were asymptomatic, 12 had clinical signs attributable to acute forms such as dysorexia and depression, weakness, low-grade fever, hypersensitivity to touch and sound, ataxia of all four limbs (4 cases) or the hind limbs (4 cases), paraparesis (6 cases) or tetraparesis (3 cases), and three subjects presented with neurological signs indicative of intracranial involvement. In most cases (10 animals) these signs were mild to moderate and

Fig. 6. Pig. *Streptococcus suis:* suppurative

meningo-encephalitis (HE, 10x).

Fig. 1. Rabbit. *Encephalitozoon cuniculi*: parassitic granuloma in the hippocampus (HE, 20x).

Fig. 2. Rabbit. *Encephalitozoon* cuniculi: parassitic antigen within a granuloma reaction (IHC, 40x).

handling, dysphagia, recumbency and death. The histological lesions showed a nonsuppurative meningo-encephalitis characterized by microabscesses (Fig. 4) and perivascular cuffings of lymphocytes and monocytes, distributed from the obex to the thalamus. The most severely affected areas were the pons and the midbrain, where often variable degree of haemorrhage was often observed. When fresh or frozen tissue (26 cases) was available, *Listeria monocytogenes* was isolated by cultural examination; moreover, IHC analysis showed the bacterial antigen predominantly in the microabscesses, less frequently in the perivascular cuffs (Fig. 5).

Fig. 3. Rabbit. *Encephalitozoon* cuniculi: parasites within a cerebral granuloma (ZN, 40x).

Fig. 4. Cow. *Listeria monocytogenes*: microabscess in the brain stem (HE, 40x).

**Streptococcosis** - In 26 (7.5%) pigs, a bacterial meningo-encephalitis was caused by *Streptococcus suis*. All our cases involved fattening piglets, 2 to 6 months of age, presenting with depression, anorexia, weight loss, ataxic gait, opisthotonus, and pedalling. The histopathological lesions showed a suppurative meningitis or mild to severe meningoencephalitis (Fig. 6), diffuse lympho-histiocytic perivascular cuffing with some neutrophils, vasculitis, neuronal degeneration and haemorrhage; sometimes the lesions extended to the choroid plexus. In 14 cases, the bacterium was isolated directly from the CNS, and in 6 of these, also from other organs such as spleen, kidney, liver, lungs and trachea. Moreover, in

handling, dysphagia, recumbency and death. The histological lesions showed a nonsuppurative meningo-encephalitis characterized by microabscesses (Fig. 4) and perivascular cuffings of lymphocytes and monocytes, distributed from the obex to the thalamus. The most severely affected areas were the pons and the midbrain, where often variable degree of haemorrhage was often observed. When fresh or frozen tissue (26 cases) was available, *Listeria monocytogenes* was isolated by cultural examination; moreover, IHC analysis showed the bacterial antigen predominantly in the microabscesses, less frequently

**Streptococcosis** - In 26 (7.5%) pigs, a bacterial meningo-encephalitis was caused by *Streptococcus suis*. All our cases involved fattening piglets, 2 to 6 months of age, presenting with depression, anorexia, weight loss, ataxic gait, opisthotonus, and pedalling. The histopathological lesions showed a suppurative meningitis or mild to severe meningoencephalitis (Fig. 6), diffuse lympho-histiocytic perivascular cuffing with some neutrophils, vasculitis, neuronal degeneration and haemorrhage; sometimes the lesions extended to the choroid plexus. In 14 cases, the bacterium was isolated directly from the CNS, and in 6 of these, also from other organs such as spleen, kidney, liver, lungs and trachea. Moreover, in

Fig. 2. Rabbit. *Encephalitozoon* cuniculi: parassitic antigen within a granuloma

Fig. 4. Cow. *Listeria monocytogenes*: microabscess in the brain stem (HE, 40x).

reaction (IHC, 40x).

Fig. 1. Rabbit. *Encephalitozoon cuniculi*: parassitic granuloma in the hippocampus

in the perivascular cuffs (Fig. 5).

Fig. 3. Rabbit. *Encephalitozoon* cuniculi: parasites within a cerebral granuloma

(HE, 20x).

(ZN, 40x).

12 cases *S. suis* was found only at the level of the trachea and lungs, but only 6 of these had neuropathological lesions. IHC confirmed *S. suis* serotype 2 infection in 10 cases (Fig. 7).

Fig. 5. Cow. *Listeria monocytogenes*: bacterial antigen within a microabscess (IHC, 40x).

Fig. 6. Pig. *Streptococcus suis:* suppurative meningo-encephalitis (HE, 10x).

**Colibacillosis** - In 21 (6%) cases, involving 15 calves, 1 lamb and 5 piglets, *E. coli* was isolated. All presented with hyperacute or acute septicaemia, depression, ataxic gait, opisthotonus and sudden death. The neuropathological lesions were similar in all cases and showed a massive suppurative meningo-encephalitis characterized by mononuclear cells and a high amount of neutrophils. There was also marked vasculitis throughout the brain; in some cases, abscess-like lesions were visible, predominantly in the brainstem. The diagnosis was confirmed by bacterial culture.

**Coenurosis -** In 17 (5 %) cases (1 cattle and 16 sheep) larvae of the *Taenia multiceps* tapeworm were identified. Clinical signs and severity of the acute phase depended on the number of eggs ingested, the extent of inflammatory response, and the location of the parasite in the CNS. The clinical records reported weight loss, apathy, adynamia, tendency to isolation, depression, blindness, head tilt and ataxia. Histologically, focal haemorrhage or malacia caused by the migratory phase of the larval stage (acute stage), were observed at the level of the cerebral hemispheres or in the caudal brainstem. In the chronic phase, the visible cysts were surrounded by atrophic tissue. The diameter of the cysts, single or multiple, varied from 1 to 4.5 cm and appeared as an empty space surrounded by a granulomatous reaction composed of macrophages, giant cells, lymphocytes and plasma cells, and a few granulocytes. Outwardly, many macrophages, more or less extensive haemorrhage, fibrin or fibrin-purulent leptomeningitis and mononuclear perivascular cuffs were sometimes observed. In many cases it was possible to extract the parasitic cysts in which the cephalic protoscolices were visible.

**West Nile encephalitis** - West Nile virus was isolated in 15 (4%) horses. The horses were 9 females and 6 males, 2 to 19 years of age, mostly trotters from the provinces of Ferrara and Bologna. The horses underwent detailed neurological examination, blood-biochemical and serological tests. The blood-biochemical tests showed no significant changes, except in 5 horses in which there were slight alterations of inflammatory proteins in the acute phase. Three horses were asymptomatic, 12 had clinical signs attributable to acute forms such as dysorexia and depression, weakness, low-grade fever, hypersensitivity to touch and sound, ataxia of all four limbs (4 cases) or the hind limbs (4 cases), paraparesis (6 cases) or tetraparesis (3 cases), and three subjects presented with neurological signs indicative of intracranial involvement. In most cases (10 animals) these signs were mild to moderate and

Zoonoses Surveillance in Italy (2000-2009):

Fig. 9. Dog**.** *Neospora caninum*: granulomatous inflammation and protozoan cysts in cerebellum (HE, 0x).

11-12).

was confirmed by electron microscopy and IHC (Figs. 9-10).

Investigation on Animals with Neurological Symptoms 39

lesions were more severe, with necrosis, cavitation and the presence of gitter cells and haemosiderin pigment within macrophages. This nonsuppurative inflammation also extended to the subpial neuroparenchyma of the medulla oblongata and pons. The other brain areas were affected less frequently. Clusters of tachyzoites or protozoal cysts were found scattered within the lesions and sometimes in normal nervous tissue. The diagnosis

> Fig. 10. Dog**.** *Neospora caninum:* protozoan cysts react with specific antigen in cerebellum

(IHC, 40x).

**Toxoplasmosis** - In 7 animals (2 dogs, 1 cat, 2 pigs and 2 dolphins) (2%) *Toxoplasma gondii* was isolated in the CNS. In the dogs, reported signs were fever, paralysis, vomiting and diarrhoea; the cat had nonspecific signs such as depression, anorexia and fever, vomiting, diarrhoea, prostration, swollen lymph nodes, ataxia, behavioural changes, and circling; the pigs showed ataxia, vomiting and diarrhoea. The histological lesions were typical of a subacute or chronic nonsuppurative meningo-encephalitis characterized by prominent perivascular cuffing of mononuclear cells and multifocal microglial nodules scattered in the surrounding neuropil. In the gray matter there were astrogliosis and mild to moderate neuronal necrosis characterized by vacuolation and chromatolysis. In the most affected areas (pons), lymphocytic vasculitis associated with haemorrhage, oedema and plasmorrhagia was predominant. Single or multiple roundish, basophilic toxoplasma cysts were found scattered in the tissue . The diagnosis was confirmed by IHC and RT-PCR. (Figs.

**Cryptococcosis** - In 4 subjects (2 dogs and 2 cats) (1%) *Cryptococcus neoformans* was isolated. The cats presented with cutaneous nodules, nystagmus, mydriasis, seizures, and also nasal ulcers and sneezing in one case; the dogs showed only neurological symptoms, with seizures, ataxia and abnormal behaviour. The histopathological findings differed between the two species. In the dogs there was a granulomatous meningo-encephalomyelitis, the leptomeninges, brain and spinal cord showed marked multifocal granulomatous inflammatory lesions, with large numbers of macrophages, lymphocytes and plasma cells; in the cats, the inflammatory response was mild. In all cases there were numerous clusters of fungal bodies spread throughout the parenchyma, consisting of circular structures with a slightly eosinophilic core surrounded by a clear halo (capsule) lending it a typical soap bubble appearance, and some were in gemmating phase. The capsule was strongly mucicarmine and PAS positive. The gray matter showed a moderate gliosis. The diagnosis

was confirmed by staining of histological sections with mucicarmine. (Fig.13).

quickly improved with recovery in all subjects. Only two horses were euthanatized because of severe disease progression; sampling of CSF, necropsy and histological examination of the CNS were performed. The first test showed moderate, predominantly mononuclear pleocytosis, and the histopathological examination revealed lesions distributed mainly at the caudal brainstem and the thoracic and lumbar spinal cord. In detail, a mild to moderate nonsuppurative encephalomyelitis characterized by perivascular cuffs of mononuclear inflammatory cells, glial nodules and focal gliosis in the gray matter were observed. In the spinal cord the lesions were bilateral and symmetrical, involving the gray matter, particularly the lateral and ventral horns (Fig. 8). At this level, a nonsuppurative inflammation was identified sometimes associated with neuronal degeneration, gliosis, neuronophagia and occasionally neutrophils. Small glial foci involved the white matter in both the spinal cord and brainstem; oedema and mild inflammatory infiltrate characterized by mononuclear cells were observed in the meninges.

Fig. 7. Pig. *Streptococcus suis:* bacterial antigen within macrophages and neutrophils cytoplasm (IHC, 10x).

 Fig. 8. Horse. *West Nile virus:* inflammatory infiltrate of mononuclear cells in the ventral horn of spinal cord (HE, 10x).

**Neosporosis** - In 4 calves and 11 dogs (4%) the protozoan *Neospora caninum* was found. The calves were a few days old and showed ataxia and limb paralysis. Histology showed similar nonsuppurative lesions in both the peripheral and the CNS. In detail, there were nonsuppurative meningomyelitis (meningo-encephalomyelitis in one case), polyradiculoneuritis, fibrosis of the peripheral nerves and neurogenic muscle atrophy. A large infiltration of lymphocytes and plasma cells was detected in the spinal meninges and nerve emergence of all spinal cord segments, especially at the level of cervico-thoracic and lombo-sacral intumescence. The inflammatory infiltrate appeared most abundant in the ventral roots along the perivascular spaces. In the most affected areas there was multifocal leucomyelitis and protozoan aggregates morphologically attributable to tachyzoites of the genus *Neospora*. In the spinal gray matter, a diffuse gliosis and chromatolysis of motor neurons were observed. The peripheral nerves showed Wallerian degeneration and marked fibrosis. The diagnosis was confirmed by PCR.

The dogs had paralysis, muscle weakness, ataxia, opisthotonus, head tilt and dysphagia. In 9 dogs the CNS was affected with meningo-encephalitis and myelitis; the other 2 dogs presented with polymyositis and chronic axonal neuropathy. An adult dog showed a severe cerebellar necrotizing inflammation with lymphoplasmacytic leptomeningitis, oedema of the white matter, incomplete atrophy and necrosis of the folia, with extensive loss of Purkinje and granular layer cells, often replaced by reactive astrocytes. In some areas, the

quickly improved with recovery in all subjects. Only two horses were euthanatized because of severe disease progression; sampling of CSF, necropsy and histological examination of the CNS were performed. The first test showed moderate, predominantly mononuclear pleocytosis, and the histopathological examination revealed lesions distributed mainly at the caudal brainstem and the thoracic and lumbar spinal cord. In detail, a mild to moderate nonsuppurative encephalomyelitis characterized by perivascular cuffs of mononuclear inflammatory cells, glial nodules and focal gliosis in the gray matter were observed. In the spinal cord the lesions were bilateral and symmetrical, involving the gray matter, particularly the lateral and ventral horns (Fig. 8). At this level, a nonsuppurative inflammation was identified sometimes associated with neuronal degeneration, gliosis, neuronophagia and occasionally neutrophils. Small glial foci involved the white matter in both the spinal cord and brainstem; oedema and mild inflammatory infiltrate characterized

> Fig. 8. Horse. *West Nile virus:* inflammatory infiltrate of mononuclear cells in the ventral

horn of spinal cord (HE, 10x).

**Neosporosis** - In 4 calves and 11 dogs (4%) the protozoan *Neospora caninum* was found. The calves were a few days old and showed ataxia and limb paralysis. Histology showed similar nonsuppurative lesions in both the peripheral and the CNS. In detail, there were nonsuppurative meningomyelitis (meningo-encephalomyelitis in one case), polyradiculoneuritis, fibrosis of the peripheral nerves and neurogenic muscle atrophy. A large infiltration of lymphocytes and plasma cells was detected in the spinal meninges and nerve emergence of all spinal cord segments, especially at the level of cervico-thoracic and lombo-sacral intumescence. The inflammatory infiltrate appeared most abundant in the ventral roots along the perivascular spaces. In the most affected areas there was multifocal leucomyelitis and protozoan aggregates morphologically attributable to tachyzoites of the genus *Neospora*. In the spinal gray matter, a diffuse gliosis and chromatolysis of motor neurons were observed. The peripheral nerves showed Wallerian degeneration and marked

The dogs had paralysis, muscle weakness, ataxia, opisthotonus, head tilt and dysphagia. In 9 dogs the CNS was affected with meningo-encephalitis and myelitis; the other 2 dogs presented with polymyositis and chronic axonal neuropathy. An adult dog showed a severe cerebellar necrotizing inflammation with lymphoplasmacytic leptomeningitis, oedema of the white matter, incomplete atrophy and necrosis of the folia, with extensive loss of Purkinje and granular layer cells, often replaced by reactive astrocytes. In some areas, the

by mononuclear cells were observed in the meninges.

Fig. 7. Pig. *Streptococcus suis:* bacterial antigen within macrophages and neutrophils cytoplasm (IHC, 10x).

fibrosis. The diagnosis was confirmed by PCR.

lesions were more severe, with necrosis, cavitation and the presence of gitter cells and haemosiderin pigment within macrophages. This nonsuppurative inflammation also extended to the subpial neuroparenchyma of the medulla oblongata and pons. The other brain areas were affected less frequently. Clusters of tachyzoites or protozoal cysts were found scattered within the lesions and sometimes in normal nervous tissue. The diagnosis was confirmed by electron microscopy and IHC (Figs. 9-10).

Fig. 9. Dog**.** *Neospora caninum*: granulomatous inflammation and protozoan cysts in cerebellum (HE, 0x).

 Fig. 10. Dog**.** *Neospora caninum:* protozoan cysts react with specific antigen in cerebellum (IHC, 40x).

**Toxoplasmosis** - In 7 animals (2 dogs, 1 cat, 2 pigs and 2 dolphins) (2%) *Toxoplasma gondii* was isolated in the CNS. In the dogs, reported signs were fever, paralysis, vomiting and diarrhoea; the cat had nonspecific signs such as depression, anorexia and fever, vomiting, diarrhoea, prostration, swollen lymph nodes, ataxia, behavioural changes, and circling; the pigs showed ataxia, vomiting and diarrhoea. The histological lesions were typical of a subacute or chronic nonsuppurative meningo-encephalitis characterized by prominent perivascular cuffing of mononuclear cells and multifocal microglial nodules scattered in the surrounding neuropil. In the gray matter there were astrogliosis and mild to moderate neuronal necrosis characterized by vacuolation and chromatolysis. In the most affected areas (pons), lymphocytic vasculitis associated with haemorrhage, oedema and plasmorrhagia was predominant. Single or multiple roundish, basophilic toxoplasma cysts were found scattered in the tissue . The diagnosis was confirmed by IHC and RT-PCR. (Figs. 11-12).

**Cryptococcosis** - In 4 subjects (2 dogs and 2 cats) (1%) *Cryptococcus neoformans* was isolated. The cats presented with cutaneous nodules, nystagmus, mydriasis, seizures, and also nasal ulcers and sneezing in one case; the dogs showed only neurological symptoms, with seizures, ataxia and abnormal behaviour. The histopathological findings differed between the two species. In the dogs there was a granulomatous meningo-encephalomyelitis, the leptomeninges, brain and spinal cord showed marked multifocal granulomatous inflammatory lesions, with large numbers of macrophages, lymphocytes and plasma cells; in the cats, the inflammatory response was mild. In all cases there were numerous clusters of fungal bodies spread throughout the parenchyma, consisting of circular structures with a slightly eosinophilic core surrounded by a clear halo (capsule) lending it a typical soap bubble appearance, and some were in gemmating phase. The capsule was strongly mucicarmine and PAS positive. The gray matter showed a moderate gliosis. The diagnosis was confirmed by staining of histological sections with mucicarmine. (Fig.13).

Zoonoses Surveillance in Italy (2000-2009):

Fig. 15. Horse. *Halicephalobus gingivalis*: perivascular granulomatous infiltrate with presence of nematode around an arteriola

From Mandara et al. Neuropatologia e neuroimmaging , Poletto ed., 2011.

confirmed by real-time PCR.

Fig. 17. Cow. *Clamydia spp.*: perivascular cuffs of mononuclear cells (HE, 20x)

(arrows) (HE, 40x)

was confirmed by real-time PCR and nested PCR.

Investigation on Animals with Neurological Symptoms 41

showed a severe nonsuppurative polioencephalomyelitis with perivascular cuffs of lymphocytes particularly around small-calibre veins of the frontal and temporal cortex, hippocampus, amygdala, and midbrain. At the hippocampal level there were also widespread gliosis with fibrillary astrocytes and neuronal necrosis (Fig. 16). The diagnosis

**Chlamydiosis** - In a 6-year-old cattle (0.2%) bacterium of the genus *Chlamydi*a spp. was isolated. The animal presented with abnormal behaviour, muscular tremors which increased when stressed, paralysis, weight loss, decreased milk production and corneal clouding. Histology showed a diffuse inflammation characterized by prominent lymphoplasmacellular and histiocytic perivascular cuffs, associated with vasculitis with few granulocytes. In the cortex there were neuronal necrosis, neuronophagia and marked gliosis (Fig. 17). The lesions were more pronounced at the level of the pons. The diagnosis was

 Fig. 16. Sheep. *Bornavirus*: perivascular cuffs and inflammatory infiltrate of mononuclear cells in hippocampus associated with pyramidal cells necrosis (HE, 4x)

 Fig. 18. Dog. *Leishmania infantum*: inflammatory infiltrate and numerous amastigotes in the epidural adipose tissue

(HE, 40x)

Fig. 11. Dolphin. *Toxoplasma gondii:*  protozoan cysts in brain (HE,40x).

 Fig. 12. Dolphin. *Toxoplasma gondii:* protozoan cysts react with specific antigen in brain (IHC, 40x).

Fig. 13. Cat. *Criptococcus neoformans:* packed yeast bodies with soap bubble appearance in the cerebral cortex (HE,10x).

 Fig. 14. Horse. *Halicephalobus gingivalis*: tangential section of a mature female in a necrotic area. Morphology of the rhabditiform oesophagus: A: body; B: isthmus; C: bulb (HE, 100x). From Mandara et al. Neuropatologia e neuroimmaging, Poletto ed., 2011.

**Halicephalobiasis** - In 2 (0.5%) horses, *Halicephalobus gingivalis* was isolated and showed a multifocal neurological syndrome characterized by ataxia, circling, hyperexcitability alternating with depression and blindness in the left eye in one of two subjects. There was also stiffness, recurrent epistaxis and profuse sweating. Rectal temperature and clinicalbiochemistry tests were normal. The neuroanatomical site of the lesion was diagnosed at the intracranial level. Neuropathological lesions were very similar. A granulomatous meningoencephalitis extended predominantly in the cerebellum, brainstem and diencephalon and appeared to be characterized by perivascular infiltrates consisting of lymphocytes, plasma cells, numerous macrophages, multinucleated giant cells and scattered eosinophils. Parasitic elements, 10 to 15 m in diameter, often oriented around the blood vessels, were present within the inflammatory lesions in the neuroparenchyma, meninges and subarachnoid space (Figs. 14 and 15).

**Borna disease** - In two sheep (0.5%) bornavirus meningo-encephalitis was diagnosed. The animals showed anorexia, ataxia, abnormal behaviour. The neuropathological examination

40x).

**Halicephalobiasis** - In 2 (0.5%) horses, *Halicephalobus gingivalis* was isolated and showed a multifocal neurological syndrome characterized by ataxia, circling, hyperexcitability alternating with depression and blindness in the left eye in one of two subjects. There was also stiffness, recurrent epistaxis and profuse sweating. Rectal temperature and clinicalbiochemistry tests were normal. The neuroanatomical site of the lesion was diagnosed at the intracranial level. Neuropathological lesions were very similar. A granulomatous meningoencephalitis extended predominantly in the cerebellum, brainstem and diencephalon and appeared to be characterized by perivascular infiltrates consisting of lymphocytes, plasma cells, numerous macrophages, multinucleated giant cells and scattered eosinophils. Parasitic elements, 10 to 15 m in diameter, often oriented around the blood vessels, were present within the inflammatory lesions in the neuroparenchyma, meninges and subarachnoid

**Borna disease** - In two sheep (0.5%) bornavirus meningo-encephalitis was diagnosed. The animals showed anorexia, ataxia, abnormal behaviour. The neuropathological examination

 Fig. 12. Dolphin. *Toxoplasma gondii:* protozoan cysts react with specific antigen in brain (IHC,

 Fig. 14. Horse. *Halicephalobus gingivalis*: tangential section of a mature female in a necrotic area. Morphology of the rhabditiform oesophagus: A: body; B: isthmus; C: bulb (HE, 100x). From Mandara et al. Neuropatologia e

neuroimmaging, Poletto ed., 2011.

Fig. 11. Dolphin. *Toxoplasma gondii:*  protozoan cysts in brain (HE,40x).

Fig. 13. Cat. *Criptococcus neoformans:* packed yeast bodies with soap bubble appearance

in the cerebral cortex (HE,10x).

space (Figs. 14 and 15).

showed a severe nonsuppurative polioencephalomyelitis with perivascular cuffs of lymphocytes particularly around small-calibre veins of the frontal and temporal cortex, hippocampus, amygdala, and midbrain. At the hippocampal level there were also widespread gliosis with fibrillary astrocytes and neuronal necrosis (Fig. 16). The diagnosis was confirmed by real-time PCR and nested PCR.

Fig. 15. Horse. *Halicephalobus gingivalis*: perivascular granulomatous infiltrate with presence of nematode around an arteriola (arrows) (HE, 40x)

From Mandara et al. Neuropatologia e neuroimmaging , Poletto ed., 2011.

 Fig. 16. Sheep. *Bornavirus*: perivascular cuffs and inflammatory infiltrate of mononuclear cells in hippocampus associated with pyramidal cells necrosis (HE, 4x)

**Chlamydiosis** - In a 6-year-old cattle (0.2%) bacterium of the genus *Chlamydi*a spp. was isolated. The animal presented with abnormal behaviour, muscular tremors which increased when stressed, paralysis, weight loss, decreased milk production and corneal clouding. Histology showed a diffuse inflammation characterized by prominent lymphoplasmacellular and histiocytic perivascular cuffs, associated with vasculitis with few granulocytes. In the cortex there were neuronal necrosis, neuronophagia and marked gliosis (Fig. 17). The lesions were more pronounced at the level of the pons. The diagnosis was confirmed by real-time PCR.

Fig. 17. Cow. *Clamydia spp.*: perivascular cuffs of mononuclear cells (HE, 20x)

 Fig. 18. Dog. *Leishmania infantum*: inflammatory infiltrate and numerous amastigotes in the epidural adipose tissue (HE, 40x)

Zoonoses Surveillance in Italy (2000-2009):

diseases and assess their possible zoonotic risk.

(HE, 40x)

**4. Discussion** 

Investigation on Animals with Neurological Symptoms 43

Fig. 21. Dog. *Rickettsia:* mononuclear vasculitis and fribrinoid necrosis of cerebrale arteriola

In this study we investigated and analyzed the prevalence of neurological zoonoses in Italy in the decade between January 2000 and December 2009.A total of 16 different zoonotic diseases were diagnosed in 271 subjects of different animal species: 7 diseases were caused by parasites, 6 by bacteria, 2 by viruses and one case was due to achlorophilic algae. Farm animals were the species most affected; no diseases were found in birds or wild animals. In 82 (23%) cases involving dogs and cats, the disease was of **"unknown aetiology"**. Such inflammatory diseases are prevalent in the dog, with a clear preference for breed and sometimes with family involvement. In our study, the species most affected was the dog (Graph.2). These disorders may, in fact, be triggered by an as yet unidentified pathogen and are therefore to be considered "potential zoonoses" (Schwab et al., 2007). The histological lesions were often suggestive of viral infections, but no causative agent has ever been isolated so far. Both rabies and canine distemper have been suggested as possible causes, presumably as expressions of atypical infection with these viruses (Summers et al., 1995). New diagnostic techniques may be useful to identify the pathogens responsible for these

In the group of defined zoonoses, **Encephalitozoonosis**, a parasitic disease, was found in 86% of the rabbits investigated (110 out of 128) coming from different provinces in Piedmont. *Encephalitozoon cuniculi*, an obligate intracellular parasite of microsporidia, can affect a wide range of mammals: rabbit, dog, cat, horse, fox, and humans as well. In the rabbit it usually presents as a chronic asymptomatic disease; in other species, especially in immunocompromised humans, more severe infections can lead to death (Mertens et al., 1997). The transmission route is probably by ingestion of food or water contaminated by spores shed with the urine of infected rabbits. Some authors suggest a vertical transmission in lagomorphs (Giordano et al., 2005). The disease is more common in pet rabbits (37-68%) than in the wild (Kunzel et al., 2010). In our study, 104 affected rabbits belonged to the group of meat animals, and only 6 were pets. Cases of human infection by microsporidia have been reported worldwide, but the natural reservoirs and modes of transmission remain to be elucidated (Furuya, 2009). *E. cuniculi* ranks third among the microsporidia that affect humans, after *E. bieneusi* and *E. bowel,* and it is also the cause of rare disseminated microsporidiosis forms, as described in a recent case of a woman with AIDS in Italy (Tasone et al. , 2002). Many authors believe that the reported data are underestimated. Infection control in rabbit-breeding is very important and should not underestimate the risks to food-

**Leishmaniasis** - In one dog (0.2%) adult parasites of the genus *Leishmania* were isolated from a spinal granuloma in an extra-dural site compressing the spinal cord. The animal presented with lymphadenopathy and paraparesis. The lesion consisted of epidural adipose tissue infiltrated by inflammatory cells, primarily composed of macrophages, granulocytes, plasma cells and lymphocytes. In the cytoplasm of the macrophages, there was a large number of small spherical structures due to uniform amastigote forms of *Leishmania* spp. The diagnosis was confirmed by IHC (Fig. 18).

**Nocardiosis** - In one dog (0.2%) *Nocardia asteroides* was isolated. Macroscopically, there was an abscess-like lesion in the brain stem (Fig. 19). The dog showed fever, depression, difficulty walking and cranial nerve deficits. Histology revealed a piogranulomatous infiltrate consisting of neutrophils, macrophages and lymphocytes. Moreover, aggregates of club-shaped eosinophilic bacteria were found. The diagnosis was confirmed by bacterial culture.

**Protothecosis** - In one dog (0.2%) achlorophilic algae were isolated. The reported signs were gastrointestinal bleeding with chronic diarrhoea, depression, anorexia, and difficulty walking. Histology showed granulomatous lesions in the neuroparenchyma and meninges consisting of numerous macrophages, lymphocytes, plasma cells and some eosinophils. In the granulomatous foci there were numerous round bodies of variable size surrounded by a PAS positive capsule (Fig. 20), which were identified as achlorophilic algae of the genus *Prototheca*. The diagnosis was confirmed by electron microscopy.

**Rickettsiosis** - In one dog (0.2%) a microorganism of the genus *Rickettsia* spp. was isolated The reported signs were fever, depression, lethargy, tetraparesis, ataxia, hyperesthesia, vestibular syndrome, seizures, and petechiae in the mucous membranes. Microscopically, there were a nonsuppurative meningo-encephalitis and vasculitis characterized by lymphohistiocytic-type cells and plasma cells infiltrating the nervous tissue and meninges; necrotic areas were also observed (Fig. 21). In this case the diagnosis was confirmed by PCR.

 Fig. 20. Dog. *Prototheca:* numerous algae with a periodic acid-Schiff (PAS) -positive (arrows) (PAS, 40x)
