The Characterisation of Lactococcus garvieae Isolated in an Outbreak of Septicaemic Disease in Farmed Sea Bass (Dicentrarchus labrax, Linnaues 1758) in Italy.

In aquaculture, Lactococcus garvieae is a common fish pathogen that can cause significant economic losses in several fresh and saltwater species. Despite the extensive range of hosts, L. garvieae infection in sea bass (Dicentrarchus labrax) has rarely been reported. During the summer of 2023, an outbreak occurred in an inland farm in the Gulf of Follonica (Tuscany, Italy). Fish of various sizes were affected, showing apathy, inappetence, erratic swimming and eye lesions, while the mortality was low (2-3% per month). Anatomopathological examinations suggested a septicaemic infection characterised by melanosis, diffuse redness (skin and fins), paleness (gills and internal organs), haemorrhages and splenomegaly. Seventy swabs from the viscera of 14 subjects were collected and colonies similar to Streptococcus spp. grew from all the samples. Lactococcus garvieae was identified via the biochemical tests, API20STREP, MALDI-TOF, 16S rDNA and whole genome sequencing. Genetical characterisation revealed remarkable differences between this isolate and the strains previously isolated in Italian fish farms. Feed treatments with flumequine and erythromycin were ineffective. Considering the limited effects of antimicrobials, preventive measures, such as vaccination and biosecurity, should be implemented.

Retrospective study of tumors from cattle slaughtered in Lombardy (Italy): preliminary evaluation on the establishment of a bovine cancer registry.

Forty­one tumors were detected in a population of 1,649,003 cattle slaughtered in 4 abattoirs in Lombardy over a 5­year period, for an overall prevalence of 2.5 tumors per 100,000 cattle. Tumors were classified according to the WHO histological classification of tumors of domestic animals. Alimentary and hemopoietic systems were commonly affected with 9 cases each. Other affected sites were the respiratory (n = 3), urinary (n = 2), endocrine (n = 2), musculoskeletal (n = 2), nervous (n = 1), and cardiovascular (n = 1) systems. The peritoneum was affected by 6 cases, while the primary location of 3 tumors of the connective tissues and 3 metastatic carcinomas was unidentified. Liver tumors and mesotheliomas, for which environmental risk factors are well­known in humans, were common, as well as tumors typically encountered in pediatric human patients (tumors of mesenchymal tissues, pulmonary blastomas and nephroblastomas). These findings suggest the useful role of bovines as sentinel and model for human carcinogenesis. Our study indicates that the establishment of a bovine cancer registry in Lombardy is feasible considering its potential contribution to understanding the role of environmental risk factors in the genesis of tumors in animals and humans.

The Italian Network of Laboratories for Veterinary Oncology (NILOV) 2.0: Improving Knowledge on Canine Tumours.

Advances in tumour research are crucial, and comparative oncology can improve the knowledge in several ways. Dogs are not only models of specific naturally occurring tumours but can also be sentinels of environmental exposures to carcinogens, as they share the same environment with their owners. The purpose of this work was to describe the data collected by The Italian Network of Laboratories for Veterinary Oncology in the first 9 years of activity (2013-2021) and to evaluate their potential epidemiological significance. Frequencies of tumour topographies and main morphologies in dogs were described, analysed and compared, calculating age-adjusted proportional morbidity ratios and considering several risk factors (breed, sex, period and region of residence). These observations allowed us to highlight differences not only in morphology and topography of some tumours but also to formulate hypotheses on the potential role of some risk factors, e.g., neutering/spaying or geographical location. In our opinion, the results of this case series confirm the importance of initiating and consolidating animal cancer registration initiatives that would facilitate the possibility of conducting multicentric collaborative studies to deepen the knowledge of the epidemiology of tumours in dogs from a comparative perspective.

Mycobacterium microti Infection in Wild Boar (Sus scrofa): Histopathology Analysis Suggests Containment of the Infection.

The European wild boar (WB) (Sus scrofa) population has rapidly expanded over the years, raising public health concerns over the species reservoir of several pathogens, including Mycobacterium microti (Mm), a Mycobacterium tuberculosis complex member. In this study, we aimed to investigate the Mm natural infection in WB in Lombardy and Emilia Romagna Italian regions by statistically evaluating the granulomatous lesions' histological features and Mm microbiological isolation. We analyzed 103 WB retropharyngeal and submandibular lymph nodes (LNs) for Mm identified by gyrB PCR-restriction fragment length polymorphism, and were retrospectively selected and histologically assessed. For each sample, Hematoxylin-eosin and Ziehl-Neelsen stained slides were evaluated. Considered histological variables were: the number of granulomas, size and maturational stage of granulomas, granulomas completeness within the section, number of multinucleated giant macrophages (MGMs), and acid-fast (AF) bacilli per granuloma. Furthermore, Mm microbiological results were also considered. Mm microbiological isolation was negatively influenced by granulomas maturation and positively affected by AF bacilli's presence within the section. Granuloma maturation was positively influenced by granuloma size and granuloma incompleteness and negatively affected by the number of granulomas in the section and the number of MGMs within the granuloma. The results indicate that granuloma maturation should ensures an efficient containment of Mm infection in the WB, suggesting that the intra-species transmission of the disease might be an unlikely event.

Natural distemper infection in stone martens (Martes foina): From infection to neutralizing antibodies.

We report an outbreak of canine distemper virus (CDV) among stone martens (Martes foina) in Italy. After being rescued in Northern Italy between April and June 2018, six subjects were kept in a wildlife and exotic animal rescue center in Bologna province. Subjects have been monitored for 15 months in captivity. Within this time-lapse, two subjects died, while among the remaining four, only one showed clinical symptoms referable to distemper. Surviving subjects have been regularly tested for CDV by means of reverse transcriptase-PCR from conjunctival and oropharyngeal swabs for eleven months. The identified viruses belonged to the Wildlife-Europe CDV genetic subgroup. Neutralizing antibodies were detected at the end of the eleven months, when all subjects tested reverse transcriptase-PCR negative. Our findings confirm the circulation of the Wildlife-Europe CDV genetic subgroup (Europe 1/South America 1 lineage) within the Italian wildlife, and improve knowledge on viral infection in stone martens.

Canine Distemper Outbreaks in Wild Carnivores in Northern Italy.

Canine distemper (CD) is a fatal, highly contagious disease of wild and domestic carnivores. In the Alpine territory, several outbreaks have occurred in the past few decades within wild populations. This study investigated the presence of canine distemper virus (CDV) infections in wild carnivores in Lombardy, relating to the different circulating genotypes. From 2018 to 2020, foxes, badgers, and martens collected during passive surveillance were subjected to necropsy and histological examination, showing classical signs and microscopic lesions related to CDV. Pools of viscera from each animal were analysed by molecular methods and immunoelectron microscopy. Total prevalences of 39.7%, 52.6%, and 14.3% were recorded in foxes, badgers, and stone martens, respectively. A phylogenetic analysis showed that the sequences obtained belonged to the European 1 lineage and were divided into two different clades (a and b) according to the geographical conformation of alpine valleys included in the study. Clade a was related to the European outbreaks originating from Germany in 2006-2010, while clade b was closely related to the CDV sequences originating from northeastern Italy during the 2011-2018 epidemic wave. Our results suggest that CDV is currently well adapted to wild carnivores, mostly circulating with subclinical manifestations and without severe impact on the dynamics of these populations.

First Report of a Severe Outbreak of Aujeszky's Disease in Cattle in Sicily (Italy).

Aujeszky's disease in cattle is caused by Suid herpes virus 1. The natural infection has been reported worldwide in bovine species and it is related to direct and indirect contact with infected pigs, which represent the main reservoir of the virus. Here, it is reported the first documented outbreak of Aujeszky's disease in cattle in Sicily (Italy). Severe itching and nonspecific neurological symptoms were the main reported clinical signs. No characteristic gross and histological features were reported other than cutaneous lesions caused by excessive pruritus and hyperaemia, haemorrhages and inflammation in the central nervous system. Diagnosis was confirmed by real time PCR and immunohistochemistry on the nervous tissue. The route of infection remained unknown, but serological data observed in pigs living in close cohabitation with cattle revealed a circulation of a wild strain of the virus in the area. This study contributes to a better knowledge of this disease in a non-conventional host and suggests the need to increase the prophylaxis control plans in specific breeding contexts.

Epitheliotropic Infections in Wildlife Ruminants From the Central Alps and Stelvio National Park.

The mountain chain of the Alps, represents the habitat of alpine fauna where the red deer (Cervus elaphus) population is the outmost numerous, followed by the chamois (Rupicapra rupicapra) and the alpine ibex (Capra ibex) at higher altitudes. Previous reports showed the circulation of epitheliotropic viruses, belonging to the families Papillomaviridae and Poxviridae, causing skin and mucosal lesions in wild ruminants of the Stelvio National Park, situated in the area. To deepen our knowledge on the natural dynamics of the infections, a passive surveillance on all the cases of proliferative skin and mucosal lesions in wild ruminants was performed. Twenty-seven samples (11 chamois, 10 red deer and 6 ibex) collected from 2008 to 2018 were analyzed by negative staining electron microscopy, histology, and PCR followed by genome sequencing and phylogenetic analyses. Results confirmed the spread of Parapoxvirus of Red Deer in New Zealand (PVNZ) in Italy, and its ability to cause severe lesions i.e., erosions and ulcers in the mouth. We showed for the first time a PVNZ/CePV1v (C. elaphus papillomavirus 1 variant) co-infection identified in one red deer. This result supports previous evidence on the ability of papillomavirus and parapoxvirus to mutually infect the same host tissue. Interestingly two ibex and one chamois showing orf virus (OV) skin lesions were shown to be co-infected with bovine papillomavirus type 1 and 2. The presence of bovine papillomavirus, in orf virus induced lesions of chamois and ibex raises the question of its pathogenetic role in these animal species. For the first time, OV/CePV1v co-infection was demonstrated in another chamois. CePV1v is sporadically reported in red deer throughout Europe and is considered species specific, its identification in a chamois suggests its ability of cross-infecting different animal species. Poxviruses and papillomavirus have been simultaneously detected also in the skin lesions of cattle, bird and human suggesting a possible advantageous interaction between these viruses. Taken together, our findings add further information on the epidemiology and pathogenetic role of epitheliotropic viruses in wild ruminants living in the central Alps and in Stelvio National Park.

Acute lameness in a cat with disseminated mycobacteriosis.

Mycobacterium avium infection was diagnosed in an adult cat showing acute lameness of the right hind limb, enlargement of the right popliteal lymph node and two cutaneous nodular lesions of the right chest wall. Conventional radiography of the proximal tibia showed a proliferative osteolytic lesion. Cytological examination of the right popliteal lymph node and the nodular skin lesions fine needle aspiration smears, demonstrated granulomatous inflammation with many negative staining bacilli within macrophages or in smears background. The diagnosis was confirmed by Ziehl­Neelsen staining of the smears and the identification of mycobacteria was performed by microbiological and molecular methods. Histopathology performed after the necropsy revealed disseminated mycobacteriosis with granulomatous mesenteric lymphadenitis, granulomatous pneumonia, hepatitis and tibial osteomyelitis. M. avium is a well­known agent of gastro­enteric, respiratory or disseminated disease in immunocompromised cats but there are few cases reported in literature of bone involvement in systemic mycobacteriosis.

Bleaching melanin in formalin-fixed and paraffin-embedded melanoma specimens using visible light: a pilot study.

In fluorescence microscopy, light radiation can be used to bleach fluorescent molecules in formalin-fixed and paraffin-embedded (FFPE) samples, in order to increase the ratio between signal of interest and background autofluorescence. We tested if the same principle can be exploited in bright field microscopy to bleach pigmented melanoma FFPE sections together with cell morphology maintenance. After dewaxing and rehydration, serial FFPE sections of a feline diffuse iris melanoma, a canine dermal melanoma, a gray horse dermal melanoma and a swine cutaneous melanoma were irradiated with visible light for 1, 2, 3, 4 and 5 days, prior to Hematoxylin and Eosin staining. Complete bleaching was obtained after 1-day treatment in feline and swine melanomas, while 2 and 3 days were required in canine and equine neoplasms, respectively. In all treated samples, cell morphology was maintained. Photo-induced bleaching combined with immunohistochemistry was tested after a 3-day photo-treatment using five different markers. According to the literature, in all samples neoplastic cells stained positive for vimentin, S100 and PNL2, while negative for FVIII and pancytokeratin. In conclusion, visible light can be effectively exploited to bleach pigmented melanoma FFPE sections prior to perform routine histochemical and immunohistochemical stains.

Morphological and molecular characterization of four Sarcocystis spp., including Sarcocystis linearis n. sp., from roe deer (Capreolus capreolus) in Italy.

Fresh (frozen/thawed) muscle samples from four 2-12-year-old roe deer (Capreolus capreolus) from the Sondrio province in north-eastern Italy were examined under a dissecting microscope, and about 180 sarcocysts were isolated and identified to morphological type in wet mounts by light microscopy (LM). Seventy-seven of these sarcocysts were subsequently examined by molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of all isolates, as well as PCR amplification, cloning and sequencing of the complete18S ribosomal RNA (rRNA) gene of two isolates of each species found. By LM, three major sarcocyst types were recognised: spindle-shaped sarcocysts, 0.5-3 mm long, either with no clearly recognisable protrusions (thin-walled) or with finger-like protrusions (thick-walled); and slender, thread-like sarcocysts, 2-3 mm long, with hair-like protrusions. Sequencing of cox1 revealed that the sarcocysts belonged to four different species. Those with no visible protrusions either belonged to Sarcocystis gracilis (n = 24) or to a Sarcocystis taeniata-like species (n = 19), whereas those with finger- and hair-like protrusions belonged to Sarcocystis silva (n = 27) and Sarcocystis capreolicanis (n = 7), respectively. The 19 cox1 sequences of the S. taeniata-like species, comprising five haplotypes, differed from each other at 0-16 of 1038 nucleotide positions (98.5-100% identity). They differed from 25 previous cox1 sequences of S. taeniata from moose and sika deer (with 98.0-100% intraspecific identity), at 33-43 nucleotide positions (95.9-96.8% interspecific identity), and there were 20 fixed nucleotide differences between the two populations. In the phylogenetic analysis based on cox1 sequences, the two populations formed two separate monophyletic clusters. The S. taeniata-like species in roe deer was therefore considered to represent a separate species, which was named Sarcocystis linearis n. sp. At the 18S rRNA gene, however, the two species could not be clearly separated from each other. Thus, there was considerable intraspecific sequence variation in the 18S rRNA gene of S. linearis (98.1-99.9% identity between 24 sequences), which was similar both in magnitude and nature to the variation previously found in this gene of S. taeniata. The new 18S rRNA gene sequences of S. linearis shared an identity of 97.9-99.6% with those of S. taeniata (overlap between intra- and interspecific identity), and in the phylogenetic tree, sequences of the two species were interspersed. By scanning electron microscopy (SEM), the sarcocysts of S. linearis were found to possess regularly spaced, thin and narrow ribbon-like cyst wall protrusions (about 2.8-3.2 µm long, 0.3-0.4 µm wide and about 0.02-0.03 µm thick), terminating in a plate-like structure of the same thickness but with an elliptic outline (about 0.3-0.4 µm wide and 0.7-0.9 µm long). The terminal plates were connected in the middle with the band-like portion of the protrusions like the board of a seesaw (tilting board). The terminal plates of adjacent protrusions were neatly arranged in a hexagonal pattern resembling tiles on a roof. Together, they formed an outer roof-like layer facing the surrounding cytoplasm of the host cell and completely covering the band-like proximal portion of the protrusions, which overlapped and were stacked in three to four layers close to the cyst surface. The sarcocyst morphology of S. linearis was consistent with that of an unnamed Sarcocystis sp. in roe deer previously found by transmission electron microscopy in several countries, including Italy. A few sarcocysts of S. gracilis and S. silva were also examined by SEM, confirming the presence of regularly distributed, short knob-like protrusions in S. gracilis (as seen in previous SEM studies) and revealing tightly packed, erect 6-7-µm-long villus-like protrusions having regularly distributed round depressions on their surface in S. silva. The sequencing of cox1 of 7, 24 and 27 new isolates of S. capreolicanis, S. gracilis and S. silva, respectively, recovered 7, 11 and 10 new haplotypes from each of the three species and expanded our knowledge on the intraspecific sequence variation at this marker. Similarly, the study revealed a more extensive intragenomic sequence variation at the 18S rRNA gene of S. capreolicanis and S. silva than known from previous studies and confirmed a near absence of such variation in the 18S rRNA gene of S. gracilis.

Detection and molecular analysis of Pseudorabies virus strains isolated from dogs and a wild boar in Italy.

Aujeszky's disease (AD) is one of the most economically important diseases of farmed pigs. Wild boars can act as reservoirs and might represent a potential threat for domestic animals, including dogs. The aim of this study was to report the results of an AD survey based on the Pseudorabies virus (PRV) genome detection in samples of dogs clinically suspected of AD and of wild boars collected during four consecutive hunting seasons in the period 2010-2014. Genomic characterization was based on the partial gC sequence of the Italian strains and the comparison with those from domestic pigs and European PRV strains circulating in wild boars. The Italian PRV strains were mainly distributed into three different clusters and revealed two interesting findings. First, there was a clear distinction between the viral strains that were isolated from dogs used for hunting and subsequently traced back to wild boars and the strains that were isolated from working dogs and subsequently found to be closely related to domestic pigs. Second, the Italian epidemiological situation was found to be different from those of European countries in that the Italian situation was characterized by the presence of both the typical Italian clades 1 and 2 and supported by new patterns of aa deletions/insertions. Italian clade 1 included strains from hunting dogs and two Italian wild boars, and Italian clade 2 grouped with recent strains from dogs that were unable to hunt and domestic pigs that were related to one old reference strain (S66) and not included elsewhere. Molecular and phylogenetic analyses of PRV strains are therefore necessary to improve the understanding of the distribution of the PRV clusters and their evolution.