**1. Introduction**

28 Non-Flavivirus Encephalitis

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Zoonoses are defined by the World Health Organization (WHO) as "Those diseases and infections naturally transmitted between vertebrae animals and man" (WHO 1959) (Palmer et al., 1998). They may be caused by viruses, bacteria, including chlamidiae and rickettsiae, fungi, protozoa, helminths and arthropods (Krauss et al., 2003), and transmitted directly (through contact with skin, hair, eggs, blood or secretions) or indirectly (by insect vectors and ingestion of contaminated food). Currently, 1415 pathogens for humans have been identified and of these approximately 61% (868) are agents of zoonoses, some of which manifest with neurological signs; 132 agents are also associated with emerging zoonoses (Asjo et al., 2007; Matassa, 2007; Taylor et al. , 2001). Neurological zoonoses are widespread, especially in the developing countries where they are not even diagnosed in most cases.

Emerging zoonoses of recently identified pathogens are Lyme disease, cryptosporidiosis, West Nile disease, transmissible spongiform encephalopathy, and possible variants of the avian influenza virus, which have found new favourable conditions for spreading. In contrast, re-emerging zoonoses are well-known diseases considered as eradicated in a given country but recur with an exponentially increasing incidence, such as tuberculosis, leptospirosis, rabies (Matassa, 2007).

Pathogens are constantly evolving and spreading in different countries through animals that act as an asymptomatic reservoir and release pathogens into the environment (Krauss et al., 2003). Among these, wild animals, both mammals and migratory birds, play an important role.

<sup>\*1</sup>Maria Domenica Pintore1, Cristiana Maurella1, Elena Bozzetta1, Carlo Cantile2, Gualtiero Gandini3, Maria Teresa Capucchio4, Arianna Calistri5, Antonio D'angelo4, Maria Caramelli1

*<sup>2</sup> Department of Animal Pathology, University of Pisa, Italy* 

*<sup>3</sup>Department of Veterinary Medical Sciences of the University of Bologna, Italy* 

*<sup>4</sup>Department of Animal Pathology, University of Turin, Italy* 

*<sup>5</sup>Department of Histology, Microbiology and Medical Biotechnologies University of Padova, Italy*

Zoonoses Surveillance in Italy (2000-2009):

Investigation on Animals with Neurological Symptoms 31

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

**cattle**

2000 to 2009

presenting with gross lesions.

**small ruminants**

microscopy after Romanonsky-type staining (Diff Quick Stain).

subjected to IHC, cultural and molecular (PCR) examination.

**rabbits**

**pigs**

**wild animals**

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

*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

*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

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

**dolphins**

**cats**

**dogs**

**horses**

**birds**

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* spp. and *Cryptococcus neoformans*.

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

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

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 and veterinary medicine essential for ensuring public health safety.

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