**2. Materials and methods**

From 01 January 2008 to 31 December 2009, 990 animals of different species presenting with neurological signs were investigated in the different participating study centres.

All subjects underwent clinical examination and only cases with central nervous system (CNS) involvement were analyzed further. Serum or whole blood and cerebrospinal fluid of some animals (CSF) were collected and examined. When the animals died or were slaughtered, necropsy was performed and samples were collected from the various organs and the CNS. Depending on the symptoms reported, samples from the spinal cord, muscles and nerves were also taken. The tissues were frozen for cultural and/or molecular investigations and fixed in 10% buffered formalin for histological and immunohistochemical (IHC) analysis.

Moreover, a retrospective investigation of cases recorded in the database of the participating study centres from 01/01/2000 to 31/12/2007 was performed. The case history of 570 animals dead or slaughtered, presenting with neurological signs and inflammatory lesions was examined. Among these, all the cases had already been diagnosed as affected by a zoonotic disease and those with lesions related to inflammatory diseases potentially transmissible to humans were selected.

In order to identify the pathogenic agent, suspected cases were submitted to cultural, biomolecular and IHC investigation using frozen or formalin-fixed tissue and/or previously stored CSF.

Neurological diseases include those caused by highly pathogenic neurotropic agents such as rabies viruses and opportunistic agents that may develop disease in the immunocompromised. These agents belong to the genus Rhabdovirus, Herpesvirus, Flavivirus, Alphavirus, Bornavirus and Circovirus; others are bacteria such as *Listeria monocytogenes*, *Borrelia garinii* and *Borrelia afzelii*, *Chlamydophila psittaci*, *Campylobacter jejuni*, or parasites such as *Toxoplasma gondii*, *Encephalitozoon cuniculi*, and *Halicephalobus gingivalis*. Recognized neurozoonotic agents among the fungi are *Aspergillus* spp., *Mucor* spp., *Candida*

Within the group of food-borne zoonoses, *E. coli* O157: H7 is a particularly relevant syndrome because ruminants, especially cattle, are the main reservoir of the bacterium,

At a 2004 meeting on emerging zoonoses jointly organized by the WHO, FAO and OIE, the factors that contribute to the emergence of zoonoses were carefully analyzed: greater pathogen adaptation and resistance (new strains); increased drug resistance; increased

Currently, there are about 60 (DPR 320/54 and European Directive 2003/99/EC), notifiable animal diseases in Italy, many of which are zoonoses, but this list is always likely to change. It is also worth pointing out that the existence of animal reservoirs (domestic and wild) and complex transmission mechanisms, by vector and food, make collaboration between human

The objective of this study was to investigate the presence of neurological zoonoses or zoonotic agents in Italy, and to estimate the epidemiological impact of neurological diseases potentially transmissible to humans. It is important to assess which species are at risk of disease and which are the best reservoirs for pathogens in order to understand the cause of

From 01 January 2008 to 31 December 2009, 990 animals of different species presenting with

All subjects underwent clinical examination and only cases with central nervous system (CNS) involvement were analyzed further. Serum or whole blood and cerebrospinal fluid of some animals (CSF) were collected and examined. When the animals died or were slaughtered, necropsy was performed and samples were collected from the various organs and the CNS. Depending on the symptoms reported, samples from the spinal cord, muscles and nerves were also taken. The tissues were frozen for cultural and/or molecular investigations and fixed in 10% buffered formalin for histological and immunohistochemical

Moreover, a retrospective investigation of cases recorded in the database of the participating study centres from 01/01/2000 to 31/12/2007 was performed. The case history of 570 animals dead or slaughtered, presenting with neurological signs and inflammatory lesions was examined. Among these, all the cases had already been diagnosed as affected by a zoonotic disease and those with lesions related to inflammatory diseases potentially

In order to identify the pathogenic agent, suspected cases were submitted to cultural, biomolecular and IHC investigation using frozen or formalin-fixed tissue and/or previously

neurological signs were investigated in the different participating study centres.

while milk and dairy products and meat are the vehicle of infection.

susceptibility of humans and animals; climate change and so on.

and veterinary medicine essential for ensuring public health safety.

their onset and which measures could be taken against their spread.

spp. and *Cryptococcus neoformans*.

**2. Materials and methods** 

transmissible to humans were selected.

(IHC) analysis.

stored CSF.

A total of 1560 cases were studied in the period from 2000 to 2009 (Graph. 1).

Graph. 1. Graphic representation of different species of animals studied in the period from 2000 to 2009

*Cerebrospinal fluid examination*: CSF samples were collected from the cisterna magna or lumbar level. Quantitative determination of total protein was carried out by photometric colorimetric testing, using as a normal range for total protein 0-30 mg/dl for fluid from the cisterna magna and 0-45 mg/dl from the lumbar region. Cell count was performed by blood cell count using a Fuchs-Rosenthal chamber, taking 0-5 cells/ mcl as the normal range, while the sediment obtained by cytocentrifugation (600 rpm for 10 min) was read by light microscopy after Romanonsky-type staining (Diff Quick Stain).

*Neuropathological examination*: the CNS was divided by a paramedian cut in two parts including the cerebral hemisphere, cerebellum and medulla oblongata. Coronal sections of formalin-fixed brain were made at the level of the brain stem, cerebellum, thalamus, hippocampus, and basal ganglia, including the cerebral cortex and any other areas presenting with gross lesions.

To evaluate the histological lesions, each sample was processed, embedded in paraffin, and microtome sections of about 4-5 μm were prepared; the sections were stained with haematoxylin-eosin. When necessary, specific histologic stains were also carried out, e.g., Masson's trichrome, Weigert van Gieson, Congo red, Luxol fast blue-cresyl violet (for evaluation of myelin), Bielschowsky silver-impregnation (for evaluation of axons), Gram staining, Grocott, Giemsa and Good-Pasture, periodic acid-Schiff's reagent (PAS) and Gomori trichrome, methenamine silver (for the recognition of fungal elements), mucicarmine (for recognition of *Cryptococcus neoformans*) and Ziehl Neelsen (for recognition of protozoan elements), as described in standard protocols (S. Daniel and T. Zanin, 1997).

When the histological investigation did not allow a definite diagnosis, samples were subjected to IHC, cultural and molecular (PCR) examination.

Zoonoses Surveillance in Italy (2000-2009):

and observed by light microscopy.

were detected in 271 animals of different species (Table 1).

confirming the diagnosis (Figs. 2 and 3).

**3. Results** 

Investigation on Animals with Neurological Symptoms 33

or fungi typing was carried out by bi-directional sequencing of amplicons using the BigDye1 Terminator v3.1 Cycle Sequencing Kit on a 3100 Genetic Analyzer (Applied Biosystems). After alignment with the SeqScape v2.5 software (Applied Biosystems), the sequences were

*Electron-microscopy*: in suspected cases of Leishmaniasis, Protothecosis and Neosporosis, electron microscopical investigations were carried out for the morphological identification of the aetiological agents. Selected tissues were cut into small pieces of about 3 mm3 which were post-fixed with osmium tetroxide, dehydrated with ethanol and embedded in epoxy resin. Sections of 1 micron thick were cut by ultramicrotome, stained with toluidine blue and observed by light microscopy. Selected blocks were then sectioned in ultra-thin sections,

*Muscle biopsy*: muscle biopsies were received fresh and wrapped in gauze moistened with saline solution and then were snap frozen by immersion in isopentane precooled in liquid nitrogen and stored at -80 °C. Eight μm-thick sections were stained and reacted with a standard panel of histochemical stains and enzyme reactions (H&E, PAS, ORO, Gomori

*Nerve biopsy*: nerve samples of 1 to 2 cm in length, depending on the nerve tested, were fixed in 2.5% glutaraldehyde solution in slight tension on a rigid support, to avoid artifacts of the nerve fibers. After washing in phosphate buffer, the samples were post-fixed in osmium tetroxide, dehydrated with ethanol of increasing concentration and embedded in epoxy resin. Sections of 1 micron thick were cut by ultramicrotome, stained with toluidine blue

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

**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

**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

compared with the ones available in GenBank by using the NCBI BLAST tool.

stained with uranyl acetate and lead citrate and observed by electron microscopy.

trichrome, ATPase pH 4.3 and 9.8, NADH-TR, SDH, COX, non specific esterase).

*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 on pure culture with an API Rapid system.

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

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 probe under standard amplification conditions.

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 at 72°C for 10 min.

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 or fungi typing was carried out by bi-directional sequencing of amplicons using the BigDye1 Terminator v3.1 Cycle Sequencing Kit on a 3100 Genetic Analyzer (Applied Biosystems). After alignment with the SeqScape v2.5 software (Applied Biosystems), the sequences were compared with the ones available in GenBank by using the NCBI BLAST tool.

*Electron-microscopy*: in suspected cases of Leishmaniasis, Protothecosis and Neosporosis, electron microscopical investigations were carried out for the morphological identification of the aetiological agents. Selected tissues were cut into small pieces of about 3 mm3 which were post-fixed with osmium tetroxide, dehydrated with ethanol and embedded in epoxy resin. Sections of 1 micron thick were cut by ultramicrotome, stained with toluidine blue and observed by light microscopy. Selected blocks were then sectioned in ultra-thin sections, stained with uranyl acetate and lead citrate and observed by electron microscopy.

*Muscle biopsy*: muscle biopsies were received fresh and wrapped in gauze moistened with saline solution and then were snap frozen by immersion in isopentane precooled in liquid nitrogen and stored at -80 °C. Eight μm-thick sections were stained and reacted with a standard panel of histochemical stains and enzyme reactions (H&E, PAS, ORO, Gomori trichrome, ATPase pH 4.3 and 9.8, NADH-TR, SDH, COX, non specific esterase).

*Nerve biopsy*: nerve samples of 1 to 2 cm in length, depending on the nerve tested, were fixed in 2.5% glutaraldehyde solution in slight tension on a rigid support, to avoid artifacts of the nerve fibers. After washing in phosphate buffer, the samples were post-fixed in osmium tetroxide, dehydrated with ethanol of increasing concentration and embedded in epoxy resin. Sections of 1 micron thick were cut by ultramicrotome, stained with toluidine blue and observed by light microscopy.
