Characterisation of Yersinia Enterocolitica Strains Isolated from Wildlife in the Northwestern Italian Alps.

Introduction: Yersiniosis is a zoonosis causing gastroenteritis, diarrhoea, and occasionally reactive arthritis and septicaemia. Cases are often linked to meat consumption and the most common aetiological agent is the Gram-negative bacilliform Yersinia enterocolitica bacterium. The occurrence of Yersinia spp. among wild animals has mostly been studied in wild boar, but it has seldom been in other species. Material and Methods: A total of 1,868 faecal samples from animals found dead or hunted were collected between 2015 and 2018 in the Valle d'Aosta region of the northwestern Italian Alps. Alpine ibex faecal samples were collected during a health monitoring program in 2018. Bacteria were isolated via PCR and confirmed as Y. enterocolitica biochemically. Strain antimicrobial susceptibility was tested by Kirby-Bauer disc diffusion, and the presence of virulence factors and antimicrobial resistance genes was investigated using whole-genome sequencing. Results: Yersinia enterocolitica strains of biotype 1A were detected in six faecal samples from red deer (0.93%), roe deer (0.49%) and red foxes (0.7%). Strains found in beech martens (3.57%) and Alpine ibex (2.77%) belonged to biotypes 1B and 5, respectively and harboured the pYPTS01 plasmid that had only been detected in Y. pseudotuberculosis PB1/+. All the isolates were resistant to ampicillin and erythromycin. Conclusion: The biovar 1A strains exhibited different virulence factors and behaved like non-pathogenic commensals. The strain from an Alpine ibex also harboured the self-transmissible pYE854 plasmid that can mobilise itself and the pYPTS01 plasmid to other strains. The beech marten could be considered a sentinel animal for Y. enterocolitica. Phenotypic resistance may account for the ability of all the strains to resist ß-lactams.

Characterization of internal ribosome entry sites according to secondary structure analysis to classify border disease virus strains.

Applying palindromic nucleotide substitutions (PNS) method, variable loci of the internal ribosome entry site (IRES) secondary structure in the 5' untranslated region (UTR) of Border disease virus sequences were analysed allowing their allocation into ten IRES classes within the species. Sequence characteristics of Turkish and Chinese strains were highly divergent from other genogroups, indicating geographic segregation and micro-evolutive steps within the species. Observed heterogeneity in the BDV species has to be considered for potential implications on diagnostic tests, control and preventive measures.

Histological and virological findings in severe meningoencephalitis associated with border disease virus in Alpine chamois (Rupicapra rupicapra rupicapra) in Aosta Valley, Italy.

In 2015, a young female Alpine chamois (Rupicapra rupicapra rupicapra), originated from the Aosta Valley Region, Northernwestern Italy, was conferred to the National Reference Centre for Wild Animal Diseases for pathologic examinations. Histological analysis revealed a severe meningoencephalitis characterized by lymphocytic and plasmacellular infiltration, gliosis, perivascular cuffs, and leptomeningitis at the level of brain and brain stem. Laboratory investigations included polymerase chain reaction, sequencing and characterization by phylogenetic analysis, and evaluation of the internal ribosome entry site secondary structure in the 5' untranslated region. These tests identified the pathological agent as border disease virus, a known health risk in domestic small ruminants. Genetic characteristics of the isolated strains, closely related to ovine and caprine strain sequences from neighboring regions of Piedmont, France, and Switzerland, suggested geographic segregation and micro-evolutive steps within the species.

The diagnostic activity on wild animals through the description of a model case report (caseous lymphadenitis by Corynebacterium pseudotuberculosis associated with Pasteurella spp and parasites infection in an alpine ibex - Capra ibex).

The diagnostic activity on wild animals implies a hard laboratory protocol where multiple disciplines, from biology to pathological anatomy until the biotechnological techniques, must be integrated to establish the causes of death. To demonstrate these concepts, the analytical approach adopted for an alpine ibex (Capra ibex) found dead in a mountain area of North Western Italy was described. The animal showed sub-costal and lymph nodes collections of green-yellow pus, fibrinous pleuropneumonia and catarrhal-hemorrhagic enteritis. Purulent process was ascribed to Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis or pseudotuberculosis, pleuropneumonia to Pasteurella multocida and Mannheimia haemolytica and enteritis to Mannheimia haemolytica. Parasitic bronchopneumonia in the caudal lobes of the lung, a severe enteric infestation by gastro-intestinal and pulmonary strongyles and coccidia were found. The cause of death in the studied ibex appeared to be a consequence of an association between various pathological processes, with bacteriological and parasitic etiology.

Evidence of circulation of the novel border disease virus genotype 8 in chamois.

Evidence of association between the novel putative border disease virus genotype 8 (BDV-8) and fatal disease in an Alpine chamois (Rupicapra rupicapra) is reported. Diagnostically, we also demonstrated, as already previously reported, the failure of BDV-specific primers (PDB1 and PDB2) to detect BDV-8.

A novel papillomavirus isolated from a nasal neoplasia in an Italian free-ranging chamois (Rupicapra r. rupicapra).

Most amniotes are the hosts of many, distantly related papillomaviruses (PVs). Infection by PVs can be asymptomatic, or lead instead to benign or malignant lesions. However, PVs infecting animals and associated with malignancies are still largely understudied. In the present study, we communicate the complete genome of a novel PV found in a nasal neoplasia of a free-ranging alpine chamois (Rupicapra r. rupicapra) in an Italian national park. Long-PCR and cloning approaches followed for Sanger sequencing were used to identify the first PV found in chamois. The genome of the novel virus - RrupPV1 - of 7256 bp in length, presents the classical PV structure, and lacks the interE2-L2 region that hosts the E5 gene in AlphaPVs and in DeltaPVs. The nucleotide identity percentage of the L1 ORF, places RrupPV1 together with OaPV3 in the same genus. The latter is a PV isolated from a squamous cell carcinoma in sheep in Sardinia. Full-genome phylogenetic reconstructions suggest that these two viruses are sister taxa, and that both of them are very distantly related to any other known PV. Many cetartiodactyl species are infected by non-monophyletic PVs. Our results exemplify further the multiple links between the infection by certain, distantly related PVs and the development of diverse cancers in animals and highlight the need of a systematic search of oncogenic and non-oncogenic animal PVs.

Salmonella spp. and antibiotic-resistant strains in wild mammals and birds in north-western Italy from 2002 to 2010.

Salmonella is an important zoonotic pathogen of economic importance. In Europe, salmonellosis is the second food-borne infection, in Italy, Salmonella is still the major cause of food-borne outbreaks. In Europe, there are many Salmonella surveillance plans on farmed animals, while Salmonella survey of wild animals is occasionally performed. The aim of this study was to investigate the presence of Salmonella including the antibiotic-resistant strains in wild animals. Between 2002 and 2010, 2,713 wild animals (canids, mustelids, birds, rodents, ungulates), were collected in north-western Italy and tested for Salmonella by classical microbiological culture method followed by serological and biochemical typing. One hundred and seventeen wild animals (63 canids, 25 mustelids, 24 birds, 5 ungulates) were found positive for Salmonella (4.3%). One hundred and thirty strains, belonging to several serotypes were isolated, and S. Typhimurium was the most common serotype found. Antibiotic susceptibility was tested by disk-diffusion test on 88 strains. Almost all the analyzed strains (97.7%) showed resistance/intermediate resistance to at least one class of antibiotics and the highest resistance values were observed for the tetracycline class. In conclusion, zoonotic and antibiotic-resistant serotypes were found in many species of wildlife.

Detection of a morphogenetically novel Sarcocystis hominis-like in the context of a prevalence study in semi-intensively bred cattle in Italy.

The aim of this study was to determine the prevalence of sarcosporidiosis in semi-intensively bred cattle in northwestern Italy. A diagnostic protocol was setup in which infected animals were identified by rapid histological examination of the esophagus, diaphragm, and heart and the detected Sarcocystis spp. were subsequently typed using conventional electron microscopy in combination with molecular techniques. Sarcosporidia cysts were detected in 78.1% of the animals and were seen most often in the esophagus. The cattle is intermediate host for Sarcocystis hominis (final host, humans and some primates), Sarcocystis cruzi (final host, domestic and wild canids), and Sarcocystis hirsuta (final host, wild and domestic cats).All these three species of Sarcocystis were identified, variously associated, with the following prevalence: S. cruzi (74.2%), S. hirsuta (1.8%), and S. hominis (42.7%). Furthermore, a new S. hominis-like (prevalence 18.5%), characterized by hook-like structures of villar protrusion and a different sequence of the 18S rRNA gene, was identified. The cattle sheds testing positive for zoonotic Sarcocystis were assessed for risk factors contributing to the maintenance of the parasite's life cycle. Significant associations emerged between consumption of raw meat by the farm owner, mountain pasturing, and absence of a sewerage system on the farm and cattle breed. Our study demonstrates that sarcosporidiosis may constitute a public health problem in Italy and indicates several issues to be addressed when planning surveillance and prevention actions. The applied diagnostic approach revealed that cattle can harbor a further type of Sarcocystis, of which life cycle and zoonotic potential should be investigated.

Characterization of Mycoplasma isolated from an ibex (Capra ibex) suffering from keratoconjunctivitis in northern Italy.

In 2005 a Mycoplasma species was isolated from ocular-conjunctival swabs from an adult male Alpine ibex (Capra ibex) from the Valle d'Aosta Region, Northern Italy. The animal suffered from bilateral ocular discharge with diffuse inflammation, severe corneal involvement of the left eye and mild corneal opacity of the right eye. Histologic examination revealed a keratoconjunctivitis characterized by lymphocytic and plasmacellular infiltration. Laboratory investigations of the isolate included culture, transmission electron microscopy, PCR, and denaturing gradient gel electrophoresis, as well as DNA sequencing of the 16S rDNA gene. These tests identified the isolate as Mycoplasma mycoides subsp. capri large-colony serovar, an organism that has occasionally been associated with keratoconjunctivitis in goats. For a correct diagnosis, it is necessary to carry out laboratory investigations, as clinical cases of keratoconjunctivitis in wild ruminants are not always ascribable to Mycoplasma conjunctivae.

Red fox (Vulpes vulpes) cannibalistic behaviour and the prevalence of Trichinella britovi in NW Italian Alps.

Food habits of the red fox (Vulpes vulpes) were studied in the Aosta Valley region (NW Italian Alps) and were related to the prevalence of Trichinella infection in the red fox itself and in two Mustelid species (the stone marten (Martes foina) and the badger (Meles meles)). The search of Trichinella by the automatic digestion of muscles samples led us to determine a prevalence of 3.5+/-1.2% in red foxes, 7.9+/-4.3% in stone martens and 1.9+/-1.8% in badgers, with no significant differences among the species. All larvae were identified as Trichinella britovi. The fox diet was assessed through the analysis of both faeces (n=180) and the stomach contents of road-killed animals (n=109). Our results confirmed the opportunistic feeding behaviour of the red fox, which is able to use various trophic resources, both of animal and vegetal origin: e.g. wild and cultivated fruits (F%=47.1; V%=67.3), rodents (F%=22.8; V%=64.8) and carrion (F%=15.6; V%=78.6) formed the bulk of the fox's diet. The frequency of occurrence of potential events of cannibalism was 1.0%, even if the complete absence of undigested remains, other than hairs, suggested the possibility of confusing cannibalism with coat-cleaning. We suggest that intra-specific necrophagy could not represent the unique way of transmission of the nematode in natural conditions.